flflfi    332 


THE 
MATURATION  OF  THE  EGG  OF  THE  MOUSE 


BY 

J.  A.  LONG  AND  E.  L.  MARK 


WASHINGTON,  D.  C. 
PUBLISHED  BY  THE  CARNEGIE  INSTITUTION  OP  WASHINGTON 

101  I 


LIBRARY 


CARNEGIE  INSTITUTION  OF  WASHINGTON,  PUBLICATION  No.  142 


CONTRIBUTIONS  FROM  ZOOLOGICAL  LABORATORY  OF  THE  MUSEUM  OF  COMPARATIVE 
ZOOLOGY  AT  HARVARD  COLLEGE.     E.  L.  MARK,  DIRECTOR.    No.  216. 


Copies  of  this  Book 
were  first  issued 

APR  3  191J 


PRESS  OF  J.  B.  LIPPINCOTT  COMPANY 
PHILADELPHIA 


CONTENTS. 


Page 

I.  Introduction i 

II.  Literature 2 

III.  Material  and  methods 6 

IV.  Time  relations  of  parturition,  maturation,  ovulation,  insemination,  and 

semination 15 

V.  Ovulation 22 

VI.  Size  of  egg 24 

VII.  Observations  on  the  maturation  processes 25 

A .  O5cy  te  I 25 

1 .  General  description  of  stages 25 

Stage  I. — Germinative  vesicle 25 

Stage  II. — Formation  of  first  maturation  spindle 26 

Stages  III-V. — Development  and  division  of   first  ma- 
turation spindle 26 

Stage  VI. — Telophase  of  first  spindle,  and  the  first  polar 

cell 27 

2.  Chromatin  parts  of  first  maturation  spindle 27 

3.  Achromatin  parts  of  first  maturation  spindle 31 

4.  Centrosomes,  circumpolar  bodies,  and  clear  region 32 

5.  Position  and  orientation  of  first  maturation  spindle 33 

6.  Abs"triction  of  first  polar  cell 34 

B.  Oocyte  II 35 

1.  General  description  of  stages 35 

Stage  VII. — Formation  of  second  maturation  spindle. .  .  35 
Stage  VIII.- — "  Equatorial  plate  "  of  second  maturation 

spindle 35 

Stage  IX. — Division  of  second  maturation  spindle 36 

Stage  X. — Telophase  of  second  spindle  and  second  polar 

cell ^ 36 

2.  Chromatin  parts  of  second  maturation  spindle 36 

3.  Achromatin  parts  of  second  maturation  spindle 38 

4.  Centrosomes,  circumpolar  bodies,  and  clear  region 39 

5.  Position  and  orientation  of  second  maturation  spindle.  ...  40 

6.  Abstriction  of  second  polar  cell 40 

C.  Ripe  egg 41 

Stage  XI. — The  pronuclei 41 

D.  Polar  cells 41 

First  polar  cell 41 

Second  polar  cell 44 

VIII.  Criticisms  and  conclusions 45 

A .  Material 45 

B.  Methods 45 

C.  Time  relations 46 

D.  Ovulation 49 

E.  Size  of  egg 50 


iv  CONTENTS. 

VI 1 1. r  Criticisms  and  conclusions — Continued.  page 

F.  Maturation  processes 51 

1 .  Germinative  vesicle 51 

2 .  First  spindle 51 

Chromatin 51 

Achromatin 55 

Centrosomes,  circumpolar  bodies,  and  clear  region 56 

Position  and  orientation 57 

Division  of  first  spindle  and  abstriction  of  first  polar  cell  59 

3 .  Second  spindle 60 

Chromatin 60 

Achromatin 62 

Centrosomes,  circumpolar  bodies,  and  clear  region 63 

Position  and  orientation 63 

Division  of  second  spindle  and  abstriction  of  second 

polar  cell 64 

4.  Polar  cells 64 

5.  Reduction 66 

IX.  Summary  of  the  principal  results  in  the  study  of  the  egg  of  the  mouse. ...  67 

Bibliography 69 

Explanation  of  plates .,-. 71 


THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 


BY  J.  A.  LONG  AND  E.  L.  MARK. 


I.  INTRODUCTION. 

Researches  into  the  maturation  phenomena  of  both  plants  and 
animals  have  been  extended  greatly  in  recent  years,  and,  although  they 
have  given  rise  to  numerous  different  and  sometimes  conflicting  theories, 
they  point  on  the  whole  toward  a  striking  uniformity  of  processes  for 
all  of  the  forms  of  life  studied.  Among  the  metazoa  investigations 
have  covered  not  only  the  maturation  of  eggs,  but  also  the  production 
of  spermatozoa.  These  investigations  have  shown  the  general  rule  to 
be  that  by  means  of  two  mitoses,  not  separated  from  each  other  by  a 
resting  nuclear  stage,  there  are  formed  in  the  one  sex  a  ripe  egg  and  two 
(or  three)  polar  cells  and  in  the  other  sex  four  spermatids.  In  many 
cases  the  origin,  structure,  and  divisions  of  the  chromosomes  involved 
in  these  mitoses  have  received  particular  attention. 

The  greater  number  of  works  on  the  maturation  divisions  of  eggs 
have  been  carried  out  on  invertebrates,  which  furnish  the  most  easily 
obtainable  material.  Work  on  vertebrates  has  been  largely  devoted  to 
the  study  of  amphibians  and  mammals.  In  the  case  of  mammals,  which 
perhaps  present  the  most  interesting  field  for  the  study  of  oogenesis, 
the  investigation  is  especially  difficult,  since  the  kinds  of  mammals 
lending  themselves  to  such  researches  are  for  several  reasons  relatively 
few;  among  these  reasons  are  the  large  size  of  the  more  common  domestic 
forms,  the  difficulty  of  breeding  wild  animals  in  captivity,  and  the 
infrequency  of  the  breeding  periods.  Of  the  mammals  most  carefully 
studied  (bat,  rabbit,  guinea-pig,  and  mouse)  the  last  has  been  believed 
to  be  the  only  exception  to  the  general  rule  that  two  polar  cells  are  formed 
in  the  maturation  of  the  egg. 

According  to  the  excellent  works  of  Tafani  and  Sobotta,  the  egg 
of  the  mouse  forms  two  polar  cells  in  only  a  small  proportion  of  cases; 
in  the  greater  proportion  of  instances  it  produces  only  one  polar  cell.  It 
was  because  of  this  apparent  exception  to  the  general  law  of  maturation 
in  metazoan  eggs  that  the  present  piece  of  work  was  undertaken.  It  was 
begun  in  1903  with  the  hope  of  finding  some  explanation  for  the  sup- 
posed two  classes  of  eggs. 

It  soon  became  clear  that  it  would  be  necessary  to  go  over  the  whole 
subject  in  a  systematic  way  on  the  basis  of  the  changes  taking  place  in 
the  chromosomes.  To  do  this  thoroughly  has  involved  so  much  time 
that  it  has  not  been  possible  to  give  special  attention  to  the  cytoplasm. 


-    S£VV       ,  V    - 
2         :- \THE-MATURATION    OF    THE    EGG    OF    THE    MOUSE. 

Since  the  summer  of  1906  papers  on  this  subject  have  been  published 
by  Gerlach,  Coe  and  Kirkham,  Kirkham,  Lams  et  Doorme,  and  lastly 
by  Sobotta.  It  is  a  satisfaction  to  confirm  some  of  the  results  of  these 
investigators.  There  are,  however,  a  number  of  points  in  which  we  do 
not  agree  with  any  of  our  predecessors ;  some  of  these  are  due  to  differ- 
ences of  interpretation,  some  to  differences  of  technique,  and  others  to 
the  insufficiency  of  material  at  the  command  of  some  of  those  who  have 
preceded  us. 

A  considerable  part  of  the  expense  incurred  in  maintaining  and 
caring  for  the  mice  has  been  covered  by  a  grant  from  the  Carnegie 
Institution  of  Washington,  and  a  part  of  the  same  grant  has  been  used 
in  procuring  the  assistance  of  an  aid  to  do  part  of  the  less  important 
technical  portion  of  the  preparation  of  slides. 


II.  LITERATURE. 

It  is  not  our  intention  to  give  here  a  summary  of  the  subject  of 
the  maturation  of  the  egg  of  either  invertebrates  or  vertebrates.  The 
reader  is  referred  to  Boveri  (1892),  Riickert  (1894),  Hacker  (1899), 
Korschelt  und  Heider  (1903),  and  Gregoire  (1905)  for  excellent  general 
reviews  of  the  literature  of  the  whole  field  or  special  portions  of  it;  to 
R.  Hertwig  (1903)  for  similar  information  relative  to  vertebrates;  and 
to  Sobotta  (1895)  and  Kirkham  (19076)  for  surveys  of  the  papers  on 
mammals.  The  following  brief  account  of  the  several  works  on  the 
mouse  will  serve  as  an  introduction  to  the  results  set  forth  in  this  paper. 
More  detailed  references  will  be  made  wherever  necessary. 

The  first  to  study  the  egg  of  the  mouse  was  Bellonci  (1885).  He 
described  in  ovarian  eggs  the  spindle  and  the  chromosomes  arranged  at 
its  equator  and  considered  them  as  being  similar  to  those  of  some  inver- 
tebrates. According  to  his  account  the  first  polar  cell  and  the  second 
spindle  are  formed  while  the  egg  is  still  in  the  ovary.  The  polar  cell  he 
considered  a  true  cell  with  a  membrane. 

Tafani  (1889)  studied  both  living  and  preserved  eggs.  He  believed 
that  the  chromosomes  of  the  first  spindle,  numbering  twenty,  were 
formed  from  the  nucleolus  while  the  egg  was  in  the  ovary,  but  that  the 
division  of  the  first  spindle  and  the  formation  of  the  first  polar  cell  took 
place  after  ovulation.  He  thought  that  in  one-fifth  of  all  cases  the 
chromosomes  left  in  the  egg  after  the  formation  of  the  first  polar  cell 
produced  a  second  spindle,  while  in  the  remaining  four-fifths  they  were 
directly  transformed  into  the  female  pronucleus.  Thus,  in  his  opinion, 
in  about  one-fifth  of  the  eggs  two  polar  cells  were  produced,  while  in 
four-fifths  there  was  only  one,  the  second  polar  cell  being  in  the  latter 
suppressed.  No  explanation  of  the  cause  of  this  difference  was  offered. 
He  said  that  each  of  the  polar  cells  contained  either  a  nucleus  or  granules, 


LITERATURE.  3 

and  that  the  first  polar  cell,  though  it  could  change  its  shape  and  also 
vary  in  size,  remained  at  the  spot  where  it  was  formed. 

Holl's  paper  (1893)  dealt  with  the  formation  of  chromosomes  from 
the  nucleolus.  He  made  the  number  eighteen.  Unfortunately,  his  ma- 
terial was  so  poorly  preserved  that  his  results  are  unreliable. 

Sobotta  (1895),  wn°  studied  a  large  number  of  eggs  (1402),  stated 
that  only  one  polar  cell  was  formed  in  about  nine-tenths  of  the  eggs — 
a  larger  proportion  than  maintained  by  Tafani — while  in  the  remaining 
one-tenth  two  were  formed.  Those  eggs  which  abstrict  only  one  polar 
cell  are  set  free  from  the  ovary  in  the  stage  of  the  germinative  vesicle 
or  of  the  early  prophase  of  the  first  maturation  spindle.  This  spindle 
is  formed  from  the  germinative  vesicle  after  the  egg  reaches  the  ovi- 
duct. Just  before  the  polar  cell  is  cut  off  the  spindle  becomes  radial  in 
position. 

In  the  other  tenth  of  the  eggs  (those  forming  two  polar  cells)  a  first 
spindle  is  formed  in  the  ovary  24  hours  before  ovulation.  He  does  not 
say  how  it  is  formed,  but  emphasizes  the  fact  that  it  lies  deep  in  the 
egg  and  is  twice  as  large  as  the  spindle  of  eggs  which  produce  but  one 
polar  cell.  The  chromosomes  also  are  different  from  those  of  the  single 
spindle.  The  division  of  the  spindle  which  accompanies  the  abstriction 
of  the  polar  cell  in  the  ovary  is  only  rarely  seen.  Then  ovulation  occurs, 
and,  while  the  ovum  is  in  the  oviduct,  the  second  spindle  arises  from  the 
chromosomes  remaining  in  the  egg.  This  spindle  is  exactly  like  the 
single  spindle  of  eggs  forming  but  one  polar  cell.  Consequently,  in  those 
eggs  which  produce  a  single  polar  cell,  it  is  the  first  spindle  and  polar 
cell  that  are  suppressed,  the  polar  cell  that  is  formed  being  the  equivalent 
of  the  second  polar  cell  of  eggs  that  form  two.  In  all  spindles  the  chromo- 
somes number  twelve  and  divide  transversely.  There  are  no  centrosomes 
nor  polar  radiations. 

In  a  later  paper  Sobotta  (1899)  describes  and  figures  the  division 
of  the  first  spindle.  He  emphasizes  its  large  size  and  deep  position  in  the 
egg  and  the  infrequency  of  this  stage.  He  believes  that  the  spindle  axis 
turns  from  a  tangential  position,  and,  just  before  the  cutting  off  of  the 
polar  cell,  becomes  radial,  with  one  pole  lying  in  the  protuberance  which 
will  become  the  polar  cell.  He  further  says,  in  correction  of  his  earlier 
statement,  that  the  second  spindle  may  be  formed  immediately  before 
ovulation. 

Gerlach  (1906)  agrees  with  Tafani  and  Sobotta  that  some  eggs 
produce  one  polar  cell,  others  two;  but  in  his  opinion  the  proportions 
are  as  three  to  one.  He  describes  the  origin  of  the  first  spindle,  the 
chromosomes  (twelve  in  number),  and  the  formation  of  the  first  polar 
cell.  This  cell  and  the  second  spindle  may  be  formed  either  in  the  ovary 
or  in  the  oviduct.  Consequently  ovulation  may  occur  at  any  time  from 
the  stage  of  the  first  spindle  to  that  of  the  second.  According  to  his 
view,  eggs  in  the  oviduct  with  no  polar  cell  must  have  the  first  spindle. 


4      THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

Although,  he  says,  only  25  per  cent  of  all  the  eggs  form  two  polar 
cells,  all  form  two  spindles,  both  of  which  divide;  however,  in  those 
eggs  which  have  only  one  polar  cell,  it  is  the  second  polar  cell  which  is 
suppressed.  This  failure  of  the  second  polar  cell  to  be  formed  is  brought 
about  by  a  rapid  division  of  the  spindle.  As  a  result  the  chromosomes 
which  would  have  been  in  the  polar  cell  are  retained  in  the  egg  cyto- 
plasm, where  they  degenerate.  The  rapid  division  is,  in  turn,  a  conse- 
quence of  late  semination. 

Gerlach  finds  that  the  two  polar  cells  are  separated  by  a  varying 
distance.  This  he  explains  as  the  result  of  the  migration  of  the  second 
spindle  from  the  point  at  which  the  first  polar  cell  was  formed.  Semina- 
tion interrupts  the  migration  and  causes  the  spindle  to  divide  in  the 
position  it  may  have  reached  when  it  was  stopped,  whatever  that  position 
may  be.  He  believes  that  in  both  divisions  the  chromosomes  are  divided 
crosswise,  but  he  thinks  that,  for  theoretical  reasons,  one  of  the  divisions 
should  be  considered  longitudinal  (i.e.,  an  equation  division).  The  chro- 
mosomes of  the  first  spindle  are  tetrads,  those  of  the  second,  dyads. 
In  one  case  he  found  what  he  considered  a  centrosome.  The  first  polar 
cell  is  larger  than  the  second. 

Lams  et  Doorme  (1907)  deal  chiefly  with  the  cytoplasm.  They, 
however,  describe  both  spindles.  The  second  spindle  is  slightly  smaller 
than  the  first,  but  it  can  be  identified  only  by  the  presence  of  the  first 
polar  cell.  They  believe  that  both  spindles  divide  and  that  two  polar 
cells  are  cut  off  in  all  cases.  The  ab  strict  ion  of  the  first  polar  cell  and  the 
formation  of  the  second  spindle  from  the  chromosomes  left  in  the  egg 
take  place  in  the  ovary.  Ovulation  occurs,  then,  during  the  stage  of  the 
second  spindle.  The  second  polar  cell  is  formed  in  the  oviduct  after 
semination.  They  maintain  that  the  second  polar  cell  is  larger  than  the 
first,  also  that  the  first  degenerates.  Each  spindle  has  twelve  chromo- 
somes; centrosomes  may  exist,  though  they  are  not  regularly  present. 

Kirkham  (1907)  believes  that  in  all  eggs  two  polar  cells  are  formed, 
the  first  always  being  produced  while  the  ovum  is  in  the  ovary.  In  his 
opinion  the  first  and  second  spindles  differ  in  the  nature  of  their  chromo- 
somes, those  of  the  first  being  tetrads,  the  second,  dyads.  The  number 
of  chromosomes  is  twelve.  Centrosomes  occur  at  the  poles  of  both  spindles. 
The  first  polar  cell  is  larger  than  the  second  and  different  in  chromatin 
content.  He  assumes  that  in  most  eggs  the  first  polar  cell  is  forced 
through  the  zona  pellucida  and  is  lost. 

Melissinos  (1907),  in  his  paper  on  the  development  of  the  mouse 
makes,  in  passing,  a  few  remarks  on  maturation.  He  thinks  that  25  per 
cent  of  the  eggs  form  two  polar  cells,  and  he  places  the  number  of  chromo- 
somes at  eight.  But  his  figures  are  so  diagrammatic  and  indicate  such 
poor  fixation  of  his  material  that  not  much  weight  can  be  given  to  them. 

Since  1895  Sobotta  (1907)  has  considerably  changed  his  former  views. 
He  now  maintains  that  one-fifth  (instead  of  one-tenth)  of  the  eggs  form 


LITERATURE.  5 

two  polar  cells,  and  that  not  only  this  one-fifth,  but  all  of  the  eggs, 
produce  two  spindles.  However,  he  still  thinks  that  in  4  out  of  every  5 
eggs  the  first  spindle  does  not  divide,  but  is  metamorphosed  directly 
into  the  monaster  of  the  second  spindle,  and  that  half  of  its  chromosomes 
must  degenerate  in  the  egg.  Thus,  in  his  opinion,  the  first  polar  cell  in 
four-fifths  of  the  eggs  is  suppressed  by  the  failure  of  the  first  spindle  to 
divide.  He  thinks  that  this  conclusion  is  supported  by  the  fact  that 
the  metakinesis  of  the  first  spindle  is  only  rarely  seen.  When  the  first 
polar  cell  is  formed  it  is  cut  off  while  the  egg  is  in  the  ovary,  and  the 
second  spindle,  too,  arises  before  ovulation.  He  adds  somewhat  to  his 
previous  description  of  the  chromosomes,  the  spindles  and  their  divisions. 
His  view  has  changed  also  in  regard  to  the  number  of  chromosomes  in 
both  spindles.  He  now  counts  sixteen  instead  of  twelve.  Sobotta  reviews 
and  criticizes  the  work  of  Gerlach,  and  touches  on  the  papers  of  Kirk- 
ham  and  Lams  et  Doorme. 

Sobotta  (1908),  in  his  latest  paper,  gives  a  clear  summary  of  the 
present  state  of  investigation  on  the  maturation  processes,  and  points 
out  that  he  believes  the  mouse  to  be  an  exception  to  the  general  rule. 
He  then  briefly  outlines  his  own  results  and  reviews  and  criticizes  the 
recent  papers  of  Gerlach,  Melissinos,  Kirkham,  and  Lams  et  Doorme. 


6      THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

III.  MATERIAL  AND  METHODS. 

The  mice  used  at  the  beginning  of  this  work  were  received  from  the 
lot  reared  by  Professor  Castle  and  Dr.  G.  M.  Allen  in  connection  with 
Dr.  Allen's  work  on  the  Heredity  of  Coat  Color  in  Mice.  Some  were 
white  and  some  were  hybrids  obtained  by  crossing  wild  gray  mice  (Mus 
mnsculus)  with  the  white  variety  bought  of  dealers.  There  were  a  few 
white  and  hybrid  individuals  of  less  simple  ancestry ;  also  black,  choco- 
late, and  golden  agouti  (Allen,  1904).  These  served  as  a  beginning  for 
the  subsequent  stock  of  400  to  500  kept  on  hand  for  material  during  the 
greater  part  of  the  past  five  years. 

As  the  vigor  and  fertility  of  the  stock  became  lessened  by  inbreeding, 
new  white  mice  procured  from  several  dealers  in  different  parts  of  the 
country  and  a  few  gray  mice  caught  wild  were  introduced  with  bene- 
ficial results.  Thus  the  animals  furnishing  eggs  for  study  were  of  cosmo- 
politan ancestry.  Besides  the  introduction  of  new  blood,  pains  were 
taken  to  mate  as  distantly  related  animals  as  possible  in  order  to  keep 
up  the  standard  of  the  stock.  With  the  idea  at  first  of  finding  out  whether 
there  was  any  possible  relation  between  the  number  of  polar  cells  and  the 
coat-color  inheritance,  whites  and  hybrids  were  mated  (giving  whites  and 
hybrids  in  equal  proportions) ;  but  on  finding  no  such  relation,  hybrids 
and  whites  were  paired  only  for  convenience  in  distinguishing  sex. 

As  a  supplement  to  the  account  of  the  care  of  mice  by  Dr.  Allen 
(1904),  whose  methods  the  writer  has  in  general  used,  the  following  may 
be  of  value  to  those  working  with  mice  and  rats.  Fig.  A  (plate  A)  shows 
a  modification  of  the  cage  originally  used  in  the  Harvard  Zoological 
Laboratory.  The  improvement  consists  in  making  the  lids  a  few  inches 
shorter  and  putting  the  hinges,  not  at  the  highest  part  of  the  cage,  but 
further  down  on  the  inclined  surface.  This  arrangement  greatly  decreases 
the  danger  of  pinching  under  the  lid  frightened  mice  which  have  run  up 
the  sides  to  the  top,  and,  finding  an  opening,  are  trying  to  get  out;  it 
also  facilitates  catching  the  mice  in  the  upper  corners. 

Since  water  left  injopen  dishes  soon  becomes  fouled,  use  was  made  of 
the  supply  bottle  shown  near  the  corner  of  the  left-hand  cover  in  fig.  A 
and  in  section  at  5,  fig.  D  (p.  9).  One  of  these  was  put  on  each  cage. 
It  consists  of  a  3 -ounce,  wide-mouth  bottle  fitted  with  a  rubber  stopper 
pierced  by  a  bent  glass  tube  of  about  6  mm.  inside  diameter.  The  tube 
has  its  lower  end  bent  just  enough  to  prevent  the  escape  of  water  when 
undisturbed  and  is  at  the  same  time  large  enough  and  open  enough  to 
allow  air  bubbles  to  ascend  as  the  water  is  lapped  out  of  the  free  end  by 
the  mouse.  This  device,  arranged  as  shown  in  fig.  A,  with  the  tube 
projecting  through  the  wire  mesh  into  the  cage,  insures  an  easily  acces- 
sible supply  of  clean,  fresh  water. 

Mice  thrive  well  on  rich  bread-and-milk,  oats,  and  sunflower  seed. 
They  find  an  occasional  bit  of  lettuce  a  welcome  addition. 


LONG  and  MARK-  Maturation  of  Egg  of  Mouse          \    \  ;J,j  i   VT  ^         ^J*  '/PLATE  A 


A.  Mouse  Cage.     (For  description  see  p.  6.) 

B,  C.  Suspended  mouse  cages,  with  self-recording  apparatus  to  indicate  approximately  the 

time  of  parturition  of  a  gravid  female.     (See  pp.  7-10.) 
G.  Chromosomes  of  first  maturation  spindle.     (See  pp.  28-30.) 


MATERIAL    AND    METHODS.  7 

For  distinguishing  individuals  the  system  of  holes  and  notches 
punched  in  the  ears,  used  by  Professor  Castle  and  Dr.  Allen,  was  em- 
ployed (Allen,  1904).  In  addition  to  a  book  for  serial  numbers,  sex, 
parentage,  color,  and  date  of  birth  arranged  according  to  the  serial 
numbers,  it  was  found  convenient  to  have  another  book  in  which  there 
was  devoted  to  each  cage  a  separate  sheet,  whereon  were  set  down  the 
serial  number,  sex,  and  color  of  each  of  the  mice  in  the  corresponding 
cage.  When  mice  were  transferred  from  one  cage  to  another,  corre- 
sponding records  were  made  on  each  sheet,  making  it  possible,  when 
necessary,  to  trace  a  mouse  from  one  cage  to  another,  and  to  determine 
its  matings. 

Individual  records  were  kept  of  the  breeding  females  only.  These 
records,  the  record  of  litters,  etc.,  were  made  on  paper  of  uniform  size 
perforated  for  file-binding.  To  lessen  the  possibility  of  error,  the  same 
sheets  also  served  for  all  subsequent  records  of  insemination,  killing, 
fixing,  etc.  Finally,  a  new  serial  number,  corresponding  with  the  numbei 
on  the  slides  prepared  from  the  killed  individuals,  was  also  recorded  on 
these  sheets. 

Sobotta  (1895)  states  that  under  natural  conditions  mice  breed 
most  actively  during  two  periods  in  the  year,  one  in  the  spring  (April  and 
May),  the  other  in  late  summer  and  early  autumn  (from  the  middle  of 
August  to  the  end  of  September) ;  but  that  if  kept  warm  they  breed  all 
winter.  Since  the  mice  used  in  this  work  have  been  kept  warm  and 
well  fed  at  all  times  of  the  year,  the  conditions  have  not  been  favorable 
for  determining  the  natural  breeding  seasons. 

As  previous  investigators  have  shown,  female  mice  are  in  heat  and 
ovulate  soon  after  parturition.  The  eggs  for  the  present  work  have 
been  obtained,  with  one  exception,  from  the  ovaries  and  oviducts  during 
the  first  40  hours  after  parturition. 

It  has  been  the  custom  of  the  junior  writer  to  look  over  the  stock 
of  breeding  mice  every  5  days  (5  days  being  the  average  time  before 
parturition  when  pregnancy  is  first  easily  recognizable)  to  note  pregnant 
females  and  to  remove  from  males  such  as  were  to  be  observed  and  killed. 
Apparently  it  has  been  the  habit  of  former  investigators  to  leave  the 
two  sexes  together  and  not  to  determine  with  exactness  either  the  time 
of  parturition  or  of  fertilization.  It  was  felt  from  the  first  that  a  fair 
degree  of  accuracy  in  the  determination  of  the  times  of  parturition  and 
insemination  would  be  of  great  advantage;  and,  since  it  was  found  that 
parturition  may  occur  at  any  time  during  the  24  hours  of  a  day,  it  was 
necessary  to  make  frequent  observations. 

In  order  to  increase  accuracy  in  observation  and  to  save  much  time 
during  both  day  and  night,  the  apparatus  illustrated  in  figs.  B,  C 
(plate  A),  and  D  was  planned  and  made  by  the  junior  writer.  Its  pur- 
pose was  to  serve  in  recording  automatically  the  approximate  time  of  the 
birth  of  litters.  In  this  apparatus  advantage  has  been  taken  of  the 


8       THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

fairly  constant  habit  of  mice  to  take  food  or  water  at  frequent  intervals. 
The  food  is  placed  on  a  stationary  shelf  in  the  cage,  whereas  the  nest 
and  the  floor  of  the  cage  are  made  independent  of  the  rest  of  the  cage 
and  of  each  other.  By  making  the  movements  of  the  delicately  poised 
nest  and  floor  self-recording,  the  activities  of  the  mouse  can  be  deter- 
mined. The  change  in  the  record  after  parturition  is  due  to  the  increase 
in  the  weight  of  the  nest  depending  on  the  presence  of  the  young  mice 
in  the  nest  even  when  the  mother  is  away.  The  apparatus  is  constructed 
on  the  principle  of  a  simple  balance,  the  movements  of  which  are  re- 
corded on  a  chronograph  drum  revolving  once  in  12  hours.  The  parts 
shown  in  fig.  D  at  i  and  2  constitute  a  unit  and  accommodate  one  mouse. 
The  apparatus  as  finally  perfected,  fig.  C,  has  a  capacity  of  four  mice,  all 
the  records  being  made  simultaneously  on  the  same  chronographic  drum. 
The  essentials  of  each  unit  are  shown  in  fig.  D;  i  is  a  diagrammatic 
side  view,  and  2  is  an  end  view.  Each  unit  consists  of  a  box,  fixed  in 
position,  but  having  a  movable  floor  composed  of  two  parts,  each  of 
which  is  suspended  independently  of  the  other  and  may  move  in  a 
vertical  direction.  The  box  (B),  about  15X12X10  inches,  rests  upon 
supports  as  seen  in  figs.  B  and  C.  Each  box  has  either  the  top  or  side 
made  of  wire  netting  having  quarter-inch  meshes  and  is  provided  with 
a  door  (D)  at  one  end  or  on  the  top  (see  lower  box  on  right  side  and 
upper  box  on  left  side,  fig.  C).  The  floor  is  of  thin,  light  wood  made  in 
two  separate  parts — a  central  part,  the  nest-floor  (fig.  D,  i  and  2,  NF), 
supporting  the  nest  (N),  and  a  marginal  part,  the  main  floor  (MF). 
The  two  parts  of  the  floor  are  suspended  from  the  ends  of  two  levers  or 
balance  arms  (NL,  FL),  the  opposite  ends  of  which  terminate  in  pointers 
(NP,  FP)  in  contact  with  the  revolving  drum  of  a  chronograph  (CR). 
The  levers  are  supported  on  pivot  fulcrums  at  O  O,  and  the  pointers  are 
made  of  very  thin  spring-brass  so  pointed  and  bent  as  to  scratch  the 
smoked  paper  enveloping  the  drum.  The  suspension  of  the  floors  is 
effected  by  means  of  thin  strips  of  wood  the  upper  ends  of  which  are 
attached  to  the  ends  of  cross-beams.  Each  cross-beam  in  turn  rests  on 
the  end  of  its  lever  by  means  of  a  glass-and-steel  bearing.  To  the  under 
side  of  the  middle  of  each  cross-beam  is  attached  a  piece  of  glass  (G,  fig. 
D,  i,  2,  3,  4),  which  rests  on  a  steel  knife-edge  (E)  secured  to  the  end  of 
the  lever  (NL  or  FL) .  Slipping  of  the  glass  on  the  steel  edge  is  prevented 
by  making  a  slot  (fig.  D,  3  and  4,  SL)  in  each  of  the  two  pieces  of  sheet 
zinc  (Z)  with  which  the  glass  (G)  is  held  in  place  on  the  under  side  of 
the  cross-beam,  the  knife-edge  (E)  occupying  the  slot.  To  all  the  edges 
of  each  floor  are  fastened  strips  of  light  tin  (T).  These  prevent  the 
mouse  from  easily  gnawing  out  and  also  keep  in  place  the  nest  (N,  which 
is  an  inverted  strawberry  basket)  and  the  sawdust  with  which  the  main 
floor  is  sprinkled.  To  the  floors  are  further  attached  light  wood  strips 
(S  S,  provided  with  metal  ends  for  reducing  friction) ,  which  keep  the 
floors  from  touching  each  other  or  the  box.  The  floors  and  attached 


MATERIAL    AND    METHODS.  9 

parts  are  counterbalanced  by  the  weights  (W  W) ,  which  may  be  so 
adjusted  that  the  floors  move  up  and  down  at  a  very  light  touch.  The 
extent  and  place  of  the  excursion  of  the  levers  are  controlled  by  check 
blocks,  shown  in  plate  A,  figs.  B  and  C,  attached  to  the  outside  of  the 
chronograph  box.  The  feed  dish  (FD)  is  on  a  little  shelf  attached  to  the 
inside  of  the  box,  and  thus  independent  of  the  movable  parts,  as  is  also 
the  water  bottle. 


•     ; 

s    \ 

FIG.  D. 

In  use,  the  weights  are  so  adjusted  that  the  empty  nest  (N)  with  its 
floor  (NF)  is  raised  to  its  upper  limit,  but  may  be  depressed  by  a  weight 
of  only  2  to  3  grams;  the  main  floor  (MF),  on  the  other  hand,  requires 
about  10  grams  to  depress  it. 

When,  therefore,  there  is  no  mouse  present,  both  parts  of  the  floor 
(NF  and  MF)  are  up,  and  the  pointers  (NP  FP,  fig.  E)  are  down.  If, 
under  these  conditions,  the  chronograph  drum  is  set  in  motion,  the  two 


10 


THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 


pointers  will  inscribe  lines  in  the  position  of  the  lines  between  a  and  b, 
fig.  E.  A  pregnant  female  placed  on  the  floor  (MF)  causes  its  pointer 
(FP)  to  go  up,  as  at  b.  As  long  as  she  remains  on  the  floor  the  record 
is  like  that  between  b  and  c.  When  she  enters  the  nest,  the  nest  pointer 
(NP)  goes  up  and  the  floor  pointer  (FP)  down,  the  record  being  that 
between  c  and  d.  When  she  leaves  the  nest  and  goes  directly  to  the 
food,  the  record  becomes  that  between  d  and  e,  as  at  first.  The  record 
at  e  shows  that  she  again  enters  the  nest,  but  on  her  way  jumps  to  the 
main  floor  (vertical  mark  on  line  FP).  If,  before  again  making  an  exit 
(as  she  must  for  water  and  food),  she  gives  birth  to  a  litter  of  little  ones, 
on  the  one  hand  her  weight  will  still  be  sufficient  to  depress  the  floor 
(MF) ,  as  at  /,  and  on  the  other,  the  young  will  be  heavy  enough  to  keep 
the  nest  down,  so,  that  no  matter  how  often  she  goes  in  and  out,  the  nest 
pointer  (NP)  will  make  an  unbroken  line,  the  floor  pointer  alone  making 
vertical  marks. 


rr                 ' 

\ 

\       1 

NP 

i 

i 

1 

a            b 

c 

d 

e 

fa 

FIG.  E. 

Knowing  the  time  when  the  record  began  or  ended,  it  is  an  easy 
matter  to  ascertain  the  limits  of  a  period  of  time,  of  day  or  night,  within 
which  parturition  occurred.  The  length  of  the  period — depending  on 
the  frequency  of  the  excursions  which  the  mouse  makes — may  vary 
from  about  15  minutes  to  6  hours,  but  is  usually  from  J  hour  to  2  hours. 
Table  i,  based  on  the  observation  of  147  mice,  is  interesting  as  showing 
the  degree  of  precision  of  these  observations. 

TABLE  i. — Observations. 


Length  of  period. 

ind^'dulls.                    Percentage. 

i  hour  or  less  

4  ^             "?O    6  —   1 

i  \  to  2  hours  

42            28    6  —    J         '       r  84    4  — 

2  J  to  3  hours  

*                  •          '              r  °4  --4- 

•?  7              2^2  — 

3i  to  5  hours  

20          13   6  + 

Over  6  hours  

^               20  + 

147 

From  this  it  is  seen  that  in  nearly  one-third  of  the  cases  the  period 
is  not  over  i  hour;  in  nearly  two-thirds  (about  60  per  cent)  it  is  not 
over  2  hours,  and  in  nearly  85  per  cent  it  does  not  exceed  3  hours.  In 
all  subsequent  calculations  the  middle  point  of  the  period  is  adopted 
in  each  case,  so  that  the  greatest  inaccuracy  as  to  time  can  not  exceed 
half  the  length  of  the  period,  and  assumably  will  be  on  the  average 
much  less. 


MATERIAL    AND    METHODS.  II 

At  this  point  it  is  convenient  to  define  two  terms  which  will  be  fre- 
quently used  in  the  following  pages,  viz,  "insemination"  and  "semina- 
tion." The  first  refers  only  to  the  introduction  of  the  male  sexual 
elements  into  the  genital  tracts  of  the  female  by  the  act  of  coitus  or 
otherwise.  The  second,  which  means  the  same  in  this  connection  as  the 
German  "Besamung,"  applies  to  the  access  of  the  spermatozoa  to  the 
eggs  in  the  oviduct,  the  coming  into  contact  of  the  male  and  female 
reproductive  cells.  Both  terms  are  distinct  from  "penetration"  and 
"fertilization." 

.-SP 


pIG-  pm — Glass  syringe  and  speculum,  about  three-fourths  actual  size. 

In  order  to  control  the  time  of  semination,  artificial  insemination 
has  been  used  in  nearly  all  the  cases  where  fertilized  eggs  have  been 
desired.  The  method  is  a  simple  one,  and  with  a  little  experience  the 
operation  becomes  easy.  It  may  be  performed  quickly  and  without 
the  use  of  ether,  and  apparently  produces  neither  pain  nor  injury  to 
the  mouse.  The  spermatozoa  are  obtained  from  the  vasa  deferentia  of 
a  male  killed  for  the  purpose  and  are  put  into  a  small  amount  of  tepid 
physiological  salt  solution  (0.75  per  cent  ordinary  table  salt),  in  which 
they  will  live  for  several  hours.  The  spermatozoa  from  one  male  are  suffi- 
cient for  several  females.  The  mass  of  spermatozoa  thus  diluted  is 
drawn  into  the  long,  narrow  part  of  a  glass  syringe  (S,  fig.  F),  made 
for  this  purpose.  If  the  spermatozoa  become  disseminated  in  the  salt 
solution — a  fact  easily  recognized  in  the  syringe  because  of  the  increas- 
ingly milky  appearance  of  its  contents  and  a  diminution  of  the  solid 
mass  of  spermatozoa — they  are  active  and  capable  of  fertilizing.  In 
practicing  artificial  insemination,  the  mouse  is  held  under  the  left  hand, 
being  confined  between  two  pieces  of  cotton-batting,  one  above  and  one 
below.  The  base  of  the  tail  is  grasped  between  the  first  joint  of  the  left 
thumb  and  the  metacarpal  of  the  left  forefinger. 

By  means  of  a  glass  speculum  (SP,  fig.  F)  introduced  into  the  vagina 
and  held  between  the  left  thumb  and  the  tip  of  the  left  forefinger,  it  is  easy 
to  see  the  somewhat  constricted  orifice  of  the  neck  of  the  uterus,  and  to 
introduce  into  the  uterus  by  means  of  the  syringe  operated  by  the  right 
hand  a  very  few  drops  of  the  fluid  containing  spermatozoa.  The  specu- 
lum and  syringe  are  best  kept  at  body  temperature  by  immersing  them 
in  hot  water,  taking  care  not  to  injure  the  spermatozoa.  Only  sperma- 
tozoa from  freshly  killed  males  were  used. 

There  can  be  no  doubt  that  the  eggs  fertilized  by  means  of  artificial 
insemination  are  perfectly  normal.  Artificial  insemination  is  a  common 
practice  in  the  breeding  of  horses  and  dogs,  the  offspring  produced  by 
such  methods  being  quite  sound  and  perfect  (Heape,  1897;  Iwanoff, 


12     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

1903).  Moreover,  in  cooperation  with  Professor  Castle,  the  junior  writer 
obtained  in  1904  by  the  above-described  method  a  litter  of  three  rats, 
which  have  been  used  for  breeding  purposes  in  Dr.  Castle's  experiments. 
Similar  breeding  experiments  with  mice  are  too  few  to  be  of  any  value ; 
but  eggs  of  mice  artificially  inseminated  when  compared  with  those  of 
mice  naturally  impregnated  appear  normal  in  every  respect.1 

In  all,  149  mice  have  been  artificially  inseminated,  but  as  only  85 
have  been  studied  in  detail  the  rest  unfortunately  can  not  be  included 
here.  31  of  the  85  have  furnished  eggs  which  contained  spermatozoa 
or  pronuclei.  A  further  discussion  will  be  found  on  page  20. 

Only  sound  mice,  white,  hybrid,  and  black,  have  been  used  for 
study.  They  have  been  killed  at  all  hours  of  the  day  and  night  during 
the  first  40  hours  after  parturition.  While  at  first  chloroform  was  used, 
it  was  found  to  be  quite  as  humane  and  quicker  to  stun  them  and  then 
break  their  necks  by  pinching  them  quickly  with  the  thumb  and  fore- 
finger just  behind  the  head. 

The  ovaries  with  the  oviducts  attached  were  immediately  removed 
and  fixed  for  from  20  to  60  minutes  in  the  following  modification  of 
Zenker's  fluid :  2  per  cent  corrosive  sublimate,  2  per  cent  potassium 
bichromate,  10  per  cent  glacial  acetic  acid.  The  fluid  was  made  up  in 
two  separate  solutions:  (A)  4  per  cent  bichromate,  (B)  4  per  cent  (aque- 
ous sol.)  sublimate  and  20  per  cent  acetic  acid.  When  desired  for  use, 
equal  portions  of  A  and  B  were  mixed.  After  fixation  the  ovaries  and 
oviducts  were  washed  in  several  changes  of  warm  water  until  fairly 
white,  i.e.,  from  12  to  24  hours;  then  left  in  70  per  cent  alcohol  contain- 
ing iodine  for  from  12  to  24  hours;  quickly  dehydrated,  cleared  in  xylol, 
and  embedded  in  paraffin.  This  process  gives  clear  fixation  of  ovarian 
eggs  without  shrinkage  of  eggs  or  nuclei  and  without  destroying  the 
finer  st  ucture.  Various  other  mixtures,  with  and  without  osmic  acid, 
have  not  given  satisfactory  results. 

The  whole  ovary  and  oviduct  were  cut  into  sections  8  micra  thick,  as 
thinner  sections  divide  the  nuclei  and  spindles  into  too  many  parts. 
The  sections  were  affixed  to  slides  with  albumen,  being  spread  by  the 
water  method,  and  were  stained  by  one  or  the  other  of  these  three  methods : 

(1)  with  iron  haematoxylin  followed  by  either  Congo  red  or  orange  G, 

(2)  with  Bohmer's  haematoxylin  and  Congo  red,  or  (3)  with  Mallory's 
(1905)  phosphotungstic-acid  haematoxylin.     The  first  gives  clear  out- 
lines, but  does  not  show  the  structure  of  chromosomes  well.     Bohmer's 
dye  when  used  for  24  hours  or  more  gives  excellent  results.    Mallory's, 
when  used  in  just  the  right  way,  is  the  best  of  any  of  the  stains  tried. 
The  method  employed  for  Mallory's  stain  was  as  follows:     From  water 
the  sections  were  put  into  a  constantly  agitated  solution  of  0.25  per  cent 

1  Since  the  above  was  written  the  junior  writer  has  obtained  two  litters  of 
perfectly  healthy  mice  by  artificial  insemination  performed  about  24  and  30  hours, 
respectively,  after  parturition. 


MATERIAL    AND    METHODS.  13 

potassium  permanganate  for  10  minutes;  rinsed  in  water;  transferred 
to  a  5  per  cent  solution  of  oxalic  acid  for  20  minutes;  washed  thoroughly 
in  water;  and  left  in  the  stain  for  from  18  to  36  hours.  The  process  was 
completed  by  a  final  rinsing,  rapid  dehydration,  and  mounting  in  balsam. 
The  results  given  in  the  following  pages  are  based  on  1,000  eggs 
obtained  from  147  mice.  Only  clearly  normal  eggs  have  been  used, 
those  in  the  ovaries  being  in  all  cases  in  large  ripe  or  nearly  ripe  fol- 
licles, never  in  small  or  manifestly  degenerating  ones.  Each  egg  was 
carefully  studied  with  a  Zeiss  2  mm.,  homogeneous  immersion,  apo- 
chromatic  objective  and  a  No.  12  compensating  ocular.  Sketches  and 
measurements  were  made  for  each  egg  on  separate  sheets  of  paper  of 
uniform  size  (see  page  7),  which  could  be  subsequently  arranged  as 
desired.  Tables  2  and  3,  which  will  be  referred  to  again,  show  in  a 
comparative  way  the  number  of  eggs  in  various  stages,  and  also  other 
data  to  be  discussed  later. 


THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 


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TIME    RELATIONS    OF    PARTURITION,  MATURATION,  ETC.     15 


IV.   TIME  RELATIONS  OF  PARTURITION,  MATURATION,   OVULA- 
TION,  INSEMINATION,  AND   SEMINATION. 

Parturition  may  occur  at  any  hour  of  the  day  or  night;  although, 
as  table  3  shows,  it  takes  place  more  frequently  in  the  early  morning. 
TABLE  3. — Number  of  cases  of  parturition  during  each  of  6  four-hour  periods  of  a  day. 


Period. 

No.  of  cases. 

Period. 

6 

10 
2 

6 

10 

2 

a.m. 
a.m. 
p.m. 
p.m. 
p.m. 
a.m. 

to 
to 
to 
to 
to 
to 

10  a.m. 
2  p.m. 
6  p.m. 
10  p.m. 
2  a.m. 
6  a.m. 

24 
21 

18 
23 

21 

40 

30 
24 

22 
21 

18 

32 

4  a.m.  to 
8  a.m.  to 
12  m.      to 
4  p.m.  to 
8  p.m.  to 
1  2  night  to 

8 

1  2 

8 

12 

4 

a.m. 
m. 
p.m. 
p.m. 
night, 
a.m. 

147 

1!  I47 

The  distribution  is  nearly  the  same  whether  the  periods  begin  at 
4  a.m.  or  at  6  a.m. 

The  eggs  which  mature  at  each  ovulation  average  nearly  seven,  and 
are  in  general  fairly  evenly  divided  between  the  two  ovaries.  In  the 
maturation  processes  of  the  eggs  of  each  individual  there  is  a  synchro- 
nism which  appears  tolerably  exact  when  the  adopted  stages  cover  fairly 
long  periods;  more  specifically,  in  most  cases  all  the  eggs  of  a  given  mouse 
are  in  one  or  the  other  of  the  following  stages:  with  (i)  the  germinative 
vesicle,  or  (2)  the  first  maturation  spindle,  or  (3)  the  first  polar  cell  and 
second  spindle.  It  rarely  occurs  that  the  eggs  from  one  ovary  are  very 
much  in  advance  of  those  produced  by  the  other;  in  fact,  a  marked 
difference  was  observed  in  only  two  mice,  and  in  these  the  most  widely 
separated  stages  exhibited,  on  the  one  hand,  the  germinative  vesicle, 
and  on  the  other,  the  first  polar  cell  and  second  spindle.  Between  eggs 
from  the  same  ovary  there  is  still  less  difference. 

If,  however,  the  processes  of  maturation  are  divided  into  shorter 
periods,  as  in  table  2  (p.  14),  the  synchronism  appears  less  perfect. 
Neglecting,  for  the  time,  mice  with  eggs  in  the  stage  of  the  second  spindle 
(VIII,  table  2) — a  stage  which  may  persist  for  24  hours  or  more — and 
considering  only  those  (50  in  number)  which  show  eggs  in  stages  between 
the  beginning  of  the  formation  of  the  first  spindle  and  the  abstriction  of 
the  first  polar  cell,  inclusive  (Stages  II  to  VI  inclusive),  it  was  found 
that  in  a  few  less  than  half  the  mice  (22)  each  individual  had  all  its 
eggs  in  only  one  stage  (either  Stage  I,  III,  IV,  or  VI),  while  the  other 
28  mice  had  eggs  which  fell  within  some  two  or  three  consecutive  stages 
from  Stage  I  to  Stage  VII.  In  no  individual  were  the  eggs  confined  to 
either  of  the  single  Stages  II,  V,  VII.  In  other  words,  one  or  the  other 
of  two  conditions  prevails;  either,  first,  all  the  eggs  from  a  given  mouse 
may  be  in  one  or  the  other  of  the  four  following  stages:  (I)  the  ger- 
minative vesicle,  (III)  the  first  spindle  with  the  chromosomes  not  yet 


l6     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

drawn  into  the  equatorial  plate,  (IV)  the  first  spindle  in  the  equatorial- 
plate  stage  with  or  without  circumpolar  bodies  (see  p.  33),  (VI)  the 
telophase  of  the  first  spindle  and  the  first  polar  cell  just  cut  off;  or, 
secondly,  some  of  the  eggs  may  be  in  one  stage,  some  in  another.  If, 
under  the  latter  condition,  some  eggs  show  either  (II)  the  beginning  of 
the  first  spindle  within  the  germinative  vesicle,  or  (V)  the  separation  of 
the  daughter  chromosomes  of  the  first  spindle,  or  (VII)  the  formation  of 
the  second  spindle,  others  are  sure  to  be  in  one  or  more  of  the  adjoining 
stages. 

The  conclusions  to  be  drawn  from  these  observations  are,  first, 
that  some  stages  occupy  less  time  than  others,  since,  owing  to  the  some- 
what imperfect  synchronism,  in  some  cases  all  the  eggs  fall  into  one 
stage,  whereas  in  other  cases  some  fall  into  one  stage,  others  into  another 
stage;  and,  secondly,  that  the  stages  passed  comparatively  quickly  are 
those  of  the  formation  of  the  first  spindle  (II),  of  the  dividing  of  the 
first  spindle  and  the  cutting  off  of  the  first  polar  cell  (V),  and  of  the 
formation  of  the  second  spindle  (VII).  Furthermore,  the  small  numbers 
of  eggs  in  these  three  stages  bear  out  these  conclusions.  In  a  similar 
way  it  can  be  shown  that  the  division  of  the  second  spindle  takes  place 
in  a  relatively  very  short  time. 

In  the  foregoing  considerations  Stages  IVa  and  IVb  can  not  with 
fairness  be  separated,  since  there  is  much  less  difference  between  them 
than  between  any  other  two  stages.  Also,  neither  of  them  is  rare.  Of 
the  two,  IVb  is  more  often  associated  with  other  stages. 

There  is  considerable  variation  among  mice  in  regard  to  the  time 
relation  between  the  stage  of  the  egg  and  the  interval  between  partu- 
rition and  killing.  This  variation  may  be  so  great  that  mice  killed  for 
eggs  in  the  oviduct  are  found  to  have  them  still  in  the  ovary,  and  vice 
versa.  Nevertheless,  a  detailed  study  of  this  relation  shows  a  uniformity 
sufficient  to  enable  one  to  say  about  when  certain  stages  occur,  and  to 
determine  approximately  the  time  of  ovulation.  Moreover,  in  connec- 
tion with  a  knowledge  of  the  relative  length  of  the  stages,  it  is  possible 
to  form  something  of  an  idea  of  the  rapidity  of  the  whole  process  and  of 
its  parts. 

How  long  before  ovulation  the  germinative  vesicle  presents  the 
conditions  shown  at  parturition  is  not  known.  It  may  be  weeks  or  even 
months.  But  it  is  quite  certain  that  for  several  days,  perhaps  weeks, 
before  ovulation  it  has  the  structure  which  is  found  during  the  first 
12  hours  after  parturition.  Usually  within  15  or  16  hours  after  parturi- 
tion the  vesicle  has  given  place  to  the  first  maturation  spindle.  More 
seldom  it  persists  longer,  even  up  to  20^  hours  after  parturition. 

The  earliest  first  maturation  spindle  that  we  have  observed  was 
formed  13}  hours  p.p.1;  and  the  latest  was  in  existence  at  28 J  hours  p.p. 
Since  the  formation  of  the  spindle  is  very  rapid,  it  is  probable  that  the 

1  For  sake  of  brevity  we  have  employed  for  post  partum  the  abbreviation  p.p. 


TIME    RELATIONS    OF    PARTURITION,  MATURATION,  ETC.       IJ 

first  spindle  may  arise  as  late  as  about  28  hours  p.p.  According  to  these 
observations,  then,  the  first  spindle  divides  "and  gives  rise  to  the  first 
polar  cell  not  earlier  than  13!  hours  p.p.,  nor  later  than  28  J  hours  p.p. 
This  conclusion  is  rendered  the  more  probable  by  the  observations  that 


14  15   16  17   18  19  20  21  22  23  24  25  26  27  28  29  30 


the  youngest  egg  in  which  the  first  polar  cell  was  completely  cut  off 
was  taken  from  a  female  killed  14  hours  p.p.,  and  that  the  oldest  egg  in 
which  the  formation  of  the  first  polar  cell  was  barely  completed  was  from 
an  individual  killed  27  hours  p.p. 

Very   frequently   the   second   spindle   has  been   found   completely 
formed  as  early  as  16  hours,  and  occasionally  as  early  as  14^,  p.p.    The 


1 8     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

latest  epoch  at  which  it  may  originate  depends  on  the  time  when  the 
first  polar  cell  is  formed,  which,  as  above  stated,  may  be  as  late  as  28 J 
hours  p.p.  It  can  be  found  in  unfertilized  eggs  in  a  normal  condition  up  to 
at  least  40  hours  p.p. 

The  accompanying  "curves"  (page  17)  are  given  to  illustrate  the 
ratio  between  two  classes  of  eggs:  the  "first  class"  embraces  eggs  con- 
taining the  germinative  vesicle,  or  the  first  spindle  in  any  of  its  stages 
up  to  the  recently  formed  first  polar  cell  (Stages  I  to  VI,  table  2);  the 
"second  class,"  eggs  of  all  succeeding  stages  (Stages  VII  to  XI).  A  con- 
sideration of  these  curves  reveals  some  facts,  or  at  least  probabilities, 
concerning  the  amount  of  time  required  for  certain  parts  of  the  matura- 
tion process.  In  the  upper  diagram  the  unbroken  line  represents  the 
number  of  eggs  in  the  first  class,  obtained  at  various  indicated  epochs 
(hours)  after  parturition,  and  the  dotted  line  the  number  of  eggs  in  the 
second  class  at  corresponding  epochs.  The  sum  of  the  two  curves, 
shown  by  the  dot-and-dash  line,  includes  all  the  eggs  obtained  up  to 
30  hours  p.p.  In  the  lower  diagram  the  unbroken  and  dotted  lines  give, 
respectively,  in  percentages  the  ratios  of  the  number  of  eggs  in  the  first 
and  second  classes  to  the  total  number  of  eggs;  they  are,  of  course, 
reciprocals  of  each  other. 

The  general  trend  of  the  percentage  curve  shows  from  the  1 4-hour  to 
the  1 6-hour  epochs  p.p.  a  rapid  decrease  in  the  proportion  of  eggs  of 
the  first  class  during  the  early  periods.  The  great  fluctuations  in  the 
periods  between  16  and  23  hours  and  between  26  and  28  hours  are 
probably  due  to  the  small  numbers  of  eggs  obtained  in  those  periods 
(compare  upper  diagram),  and  very  likely  would  disappear  to  a  large 
extent  with  more  abundant  material. 

The  whole  process  of  maturation  can  be  conveniently  divided  into 
two  parts,  the  first  embracing  those  stages  which  are  included  in  the 
first  class  of  eggs  and  the  second  part  those  in  the  second  class.  It 
should  be  borne  in  mind  that  eggs  of  the  first  class  are  constantly  in  a 
state  of  activity  and  are  steadily  advancing  toward  the  formation  of  the 
first  polar  cell;  whereas  eggs  of  the  second  class,  if  not  seminated,  re- 
main for  24  hours  or  more  in  a  quiescent  condition  in  the  stage  of  the" 
second  spindle  (Stage  VIII).  Consequently,  the  length  of  the  period 
in  which  the  eggs  of  the  first  class  fall  would  be  an  approximate  measure 
of  the  time  required  for  that  part  of  the  process;  but  a  similar  period 
for  the  second  class  would  not  be  a  measure  of  the  amount  of  time  nec- 
essary for  the  completion  of  the  second  part  of  the  process.  The  time 
required  for  the  latter  is  calculated  by  other  means.  Since  in  eggs  above 
the  twenty-third  hour  p.p.  the  proportion  of  the  first  class  is  very  small, 
it  can  be  said  that  usually  the  first  part  of  maturation  is  completed 
within  the  period  between  14  and  23  hours  p.p.  When  it  is  noted, 
further,  that  the  curve  representing  the  percentage  of  eggs  in  the  first 
class  drops  very  rapidly  from  the  i4th  to  the  i8th  hour,  it  is  fair  to 


TIME    RELATIONS    OF    PARTURITION,  MATURATION,  ETC.       1 9 

assume  that  the  first  part  of  maturation  in  a  large  majority  of  cases 
occurs  between  the  1 4-hour  and  the  1 8-hour  epochs  p.p.  While  it  is 
quite  possible  that  the  first  part  of  maturation  requires  fully  4  hours 
(as  for  example  from  14  to  18  hours  p.p.),  it  seems  highly  probable  that 
it  may  be  accomplished  within  2  hours,  for  the  reason  that  at  the 
1 6-hour  epoch  as  many  eggs  have  reached  the  second  part  of  matura- 
tion as  are  still  in  the  first  part.  If  that  assumption  is  true,  the  process 
beginning  at  14  hours  p.p.  would  be  finished  at  16  hours,  that  starting  at 
1 6  hours  would  end  at  18  hours,  and  so  on. 

The  second  part  of  the  maturation  process — the  formation  of  the 
second  spindle,  the  division  of  the  spindle,  and  the  formation  of  the 
second  polar  cell — probably  requires  only  a  very  short  time  (perhaps 
only  a  few  minutes).  But  the  period  when  this  takes  place  depends, 
as  Tafani  and  Sobotta  have  pointed  out,  on  the  time  of  semination, 
this  part  of  the  maturation  process  being  apparently  dependent  on  the 
stimulation  due  to  the  presence  of  the  spermatozoon  in  the  egg. 

Now,  the  earliest  stage  of  an  egg  containing  a  spermatozoon  that 
we  have  observed  came  from  a  mouse  killed  20^  hours  p.p.,  but  most  of 
the  fertilized  eggs  were  obtained  from  animals  killed  between  23  and 
31  hours  p.p.  Thus  generally  the  second  part  of  the  process  occurs  at  a 
period  which  begins  somewhere  between  2j  (20  J  minus  18)  and  17  (31 
minus  14)  hours  after  the  completion  of  the  first.  Consequently  the  whole 
process  of  maturation  probably  requires  not  less  than  4  hours.  However, 
as  we  have  seen  (p.  1 7) ,  the  first  part  of  maturation  may  occur  quite  late — 
as  late  as  28^  hours  p.p.  In  such  case  it  is  entirely  conceivable  that 
spermatozoa  might  reach  the  oviduct  simultaneously  with  the  eggs,  and, 
as  a  result,  the  second  part  of  maturation  might  not  be  delayed  but  begin 
immediately  on  the  completion  of  the  first. 

It  must  be  concluded,  then,  that  the  process  of  maturation  (i.e., 
from  the  disappearance  of  the  germinative  vesicle  to  the  completion  of 
the  second  polar  cell)  may  be  accomplished  within  about  2  hours,  but 
probably  requires  more,  from  4  to  15  hours,  the  longer  period  (above 
4  hours)  being  due  to  delay  in  the  time  of  semination. 

The  time  of  ovulation  is  not  rigidly  fixed  with  regard  either  to  par- 
turition or  to  the  maturation  of  the  egg.  Table  2  shows  the  location 
(ovary,  oviduct,  etc.)  of  eggs  in  the  several  stages,  and  table  4  the  in- 
tervals p. p.  when  eggs  in  Stages  III,  IV6,  VI,  VII,  and  VIII  were  obtained. 
Also,  table  4  does  not  include  all  the  mice  whose  eggs  fall  in  the  above 
stages,  but  only  those  bearing  on  ovulation.  Two  mice  (Stage  IV6, 
table  2  and  table  4),  one  killed  14!  and  the  other  i8J  hours  p.p.,  showed 
in  the  periovarial  space  two  eggs  and  one  egg  respectively.  Each  of  the 
three  eggs  had  the  first  spindle  in  the  "equatorial-plate"  stage  with 
circumpolar  bodies.  The  ovaries  contained  other  eggs  of  the  same 
stage  in  ripe  follicles.  Referring  again  to  tables  2  and  4,  the  eggs  in  Stage 
VI  were  all  found  in  the  ovary  except  four  (from  two  mice  killed  1 6J  and 


20 


THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 


24  hours  p.p.,  respectively),  which  were  in  the  oviduct  along  with  other 
eggs  in  Stages  VII  and  VIII.  The  eight  eggs  of  Stage  VII  which  were 
from  the  oviduct  came  from  six  mice  killed  at  from  15  to  17  hours  p.p., 
all  being  associated  with  eggs  in  Stage  VIII.  Of  the  three  eggs  of  Stage 
VII  which  were  still  in  the  ovary,  one  was  from  a  mouse  killed  22  J 
hours  p.p.,  and  two  were  from  two  mice  killed  15^  and  16  hours  p.p., 
respectively.  Of  the  eggs  in  Stage  VIII,  many  were  in  the  oviduct  even 
as  early  as  14!  hours  p.p.  Among  a  few  (7)  mice,  however,  having  all 
their  eggs  in  Stage  VIII,  in  three  (killed  14!,  19^,  and  22 J  hours  p.p., 
respectively)  eggs  occurred  in  the  ovary,  in  the  periovarial  space,  and  in 
the  oviduct;  in  two  (killed  14!  and  28  hours  p.p.,  respectively)  eggs  were 
found  in  both  ovary  and  oviduct;  in  one  (killed  14^  hours  p.p.)  eggs  were 
discovered  in  both  ovary  and  periovarial  space;  and  in  another  (killed 
1 6  hours  p.p.)  the  periovarial  space  and  oviduct  contained  eggs.  In 
Stage  III  some  eggs  were  observed  in  the  ovary  28 J  hours  p.p.  It  fol- 
lows, therefore,  that  ovulation  occurs  from  14^  to  28^  hours  p.p.,  and 
that  eggs  when  discharged  may  be  in  any  stage  from  the  end  of  the 
"equatorial-plate"  stage  of  the  first  spindle  (Stage  IV6)  to  that  of  the 
second  spindle  (Stage  VIII). 

TABLE  4. — Mice  killed  during  ovulation,  showing  location  of  eggs  and 
hours  p.p.  when  they  were  obtained. 


Stage. 

Individual  No. 
of  mouse. 

Hours  p.p. 
when  killed. 

Location. 

Ovary. 

Periovarial 
space. 

Oviduct. 

Ill 

183                          28* 

X 

IVb 

220                                  I4f 

X 

X 

140                      i8£ 

X 

X 

VI 

no                      i6£ 

X 

126                      24 

X 

VII 

75                      l6 

X 

X 

lioi                     15 

X 

103 

I5* 

X 

107 

X 

no 

16^ 

X 

144 

17 

X 

86 

16- 

X 

H 

IOO 

X 

142                                22j 

X 

VIII 

Several 

14! 

X 

70 

14! 

X 

X 

75 

16 

X 

X 

89 

14! 

X 

X 

X 

95 

14! 

X 

X 

1  13 

19^ 

X 

X 

X 

118 

22^ 

X 

X 

X 

187 

28 

X 

X 

'Eggs  in  Stage  VII  in  only  one  oviduct.     Eggs  in  Stage  I  were  also 
found,  but  in  the  ovary  of  the  opposite  side  of  the  body. 

As  already  mentioned  (p.  12),  out  of  85  mice  artificially  inseminated 
31  produced  fertilized  eggs.     No  attempt  is  made  here  to  analyze  ex- 


TIME    RELATIONS    OF    PARTURITION,  MATURATION,  ETC.       21 

haustively  the  reasons  for  failure  in  so  many  (54)  cases,  but  some  of  the 
apparent  causes  will  be  given  for  the  benefit  of  those  who  may  wish  to 
use  the  method  for  breeding  purposes,  or  for  a  continuation  of  the  study 
of  the  phenomena  of  fertilization  in  mammals. 

The  number  of  hours  after  parturition  when  mice  were  inseminated 
varied  from  9^  to  28-J;  and  the  time  between  insemination  and  killing 
varied  from  3^  to  17^  hours,  as  many  time  combinations  as  possible 
being  made.  Before  considering  the  two  classes  of  eggs  from  the  insem- 
inated individuals — the  fertilized  and  the  unfertilized — 14  cases  can  at 
once  be  deducted  from  the  latter,  because  the  eggs  in  those  14  mice  were 
found  in  the  ovaries,  where  semination  is  of  course  not  to  be  expected. 

In  the  first  class  (cases  resulting  in  fertilized  eggs)  the  times  of 
insemination  were  pretty  evenly  distributed  between  i6j  and  28 J  hours 
p.p. ;  two, however,  lay  outside  these  limits, being  at  pj  and  14^  hours  p.p., 
respectively.  All  these  mice  were  killed  at  from  4  to  13 \  hours  after 
insemination,  the  one  inseminated  at  9^  being  killed  13 \  hours  later 
(23  hours  p.p.),  and  the  one  at  14^,  6  hour^s  later  (20^  hours  p.p.)-  In 
the  second  class  (resulting  in  unfertilized  eggs)  most  of  the  insemina- 
tions were  made  between  n  and  i8j  hours  p.p.;  a  few,  however,  were 
evenly  distributed  between  20  J  and  284  hours  p.p.  The  animals  were 
killed  at  from  3^  to  17^  hours  after  insemination. 

A  comparison  of  the  two  classes  brings  out  the  fact  that  the  insemi- 
nations in  both  extend  over  almost  exactly  the  same  period  of  time,  but 
with  a  somewhat  different  distribution;  and  a  more  detailed  examina- 
tion of  the  data  (not  recorded  here)  shows  that  as  the  inseminations 
were  delayed  more  and  more  after  parturition  the  proportion  of  suc- 
cessful ones  increased.  Accordingly,  the  optimum  time  for  insemination 
lies  between  18  and  30  hours  p.p. 

The  most  obvious  causes  of  failure  are  (i)  too  early  insemination, 
in  which  case  possibly  the  conditions  of  the  uterus  are  sometimes  un- 
favorable for  the  continued  existence  of  the  spermatozoa,  (2)  killing  too 
soon  after  insemination  to  allow  the  spermatozoa  time  to  reach  the  eggs, 
(3)  late  ovulation,  and  (4)  combinations  of  two  or  all  of  these  factors. 

The  time  required  for  the  spermatozoa,  after  introduction  into  the 
uterus,  to  reach  the  eggs  in  the  first  part  of  the  oviduct  nearest  the 
ovary  varies  from  4  to  7  hours  in  mice  inseminated  about  the  same 
number  of  hours  p.p.  Of  these  eggs  some  contained  the  heads  of  sper- 
matozoa, some  both  pronuclei.  Assuming,  as  is  reasonable,  that  all  the 
eggs,  because  they  lie  very  near  one  another,  are  seminated  at  nearly 
the  same  time,  one  must  conclude  that  the  time  required  for  a  sperma- 
tozoon to  develop  into  a  pronucleus  is  very  short  indeed.  According 
to  the  same  reasoning  pronuclei  must  grow  very  rapidly.  Since  the  first 
spindle  never  persists  until  the  egg  reaches  the  oviduct,  semination  occurs 
only  during  the  stage  of  the  second  maturation  spindle.  An  account  of 
the  effect  of  semination  on  maturation  is  given  on  p.  35. 


22     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 


V.  OVULATION. 

The  time  when  ovulation  occurs  in  relation  to  parturition  and  the 
maturation  of  the  egg  has  already  been  given  (p.  19).  No  attempt  has 
been  made  to  determine  how  often  ovulation  occurs,  nor  the  regularity 
of  such  occurrence.  It  is  perhaps  worthy  of  record,  however,  that  a 
female  kept  isolated  and  killed  6  weeks  after  parturition  gave  eggs  in 
the  oviduct  in  the  stage  of  the  second  spindle,  a  fact  which  does  not 
conflict  with  Sobotta's  statement  that  ovulation  occurs  at  intervals  of  3 
weeks.  On  the  other  hand,  careful  records  of  the  births  of  litters  show 
that  the  3 -weeks  periods  are  not  constant,  in  fact,  that  they  vary  by 
several  days.  As  far  as  known  to  us,  no  careful  examination  into  the 
causes  of  these  variations  in  mice  has  been  made.1 

Although  the  irregularity  in  the  occurrence  of  ovulations — which 
may  be  as  great  as  days  or  even  weeks — may  possibly  be  caused  by 
coitus,  it  is  certain  that  the  first  ovulation  after  parturition  is  entirely 
independent  of  such  external  condition,  because  females  removed  from 
males  before  they  give  birth  to  young  always  furnish  eggs  in  the  oviduct 
if  killed  at  the  proper  time. 

Just  as  there  is  a  lack  of  perfect  synchronism  in  the  maturation 
processes,  so  here  some  eggs  pass  from  the  ovary  early  enough  to  have 
already  reached  the  oviduct,  while  in  the  same  individual  others  are  in 
the  periovarial  space,  and  still  others  are  in  the  ovary.  Since  in  each  of 
seven  cases  eggs  were  found  in  two,  or  sometimes  three,  of  these  places, 
it  is  highly  probable  that  in  few  or  no  mice  do  the  eggs  leave  the  follicles 
at  exactly  the  same  time. 

In  the  ovaries  of  a  mouse  killed  22  J  hours  p.p.,  there  occurred  three 
follicles  (plate  6,  figs.  38,  39,  40)  showing  in  a  rare  way  three  stages  in 
the  process  of  ovulation.  First,  the  completely  ripe  follicle  about  to 
rupture  (fig.  38) ;  secondly,  the  ruptured  follicle  before  the  escape  of  the 
egg  (fig.  39) ;  and,  thirdly,  the  flowing  out  of  the  contents  of  the  follicle 
carrying  the  egg  with  them  (fig.  40) .  They  are  all  later  conditions  than 
those  figured  by  Sobotta  (1907),  and  are  an  interesting  supplement  to 
his  observations.  In  fig.  38  the  granulosa  cells  which  form  the  sides 
and  fundus  of  the  follicle  are  so  numerous  that  they  form  a  thick  wall 
several  (four  or  more)  cells  deep,  as  Sobotta  has  pointed  out;  but  the 
side  of  the  follicle  next  the  surface  of  the  ovary  has  already  become 
attenuated  to  such  an  extent  that  at  its  middle  the  nuclei  of  granulosa 
cells  are  entirely  wanting.  The  theca  folliculi  having  also  disappeared 
in  that  region,  the  fluid  contents  of  the  follicle  come  into  direct  contact 
with  the  germinal  epithelium,  which  is  stretched  out  into  a  thin  mem- 

1  POSTSCRIPT. — During  the  year  1910  Dr.  J.  Frank  Daniel  has  independently 
found  the  variation  in  the  gestation  of  mice  to  be  even  greater  than  we  have  stated. 
He  has  worked  this  out  in  considerable  detail,  as  may  be  seen  in  his  forthcoming 
paper  in  the  Journal  of  Experimental  Zoology,  Vol.  9,  No.  4. 


OVULATION.  23 

brane  with  widely  scattered  nuclei.  The  discus  proligerus  is  already 
separated  from  the  rest  of  the  granulosa,  and  its  cells,  except  those  con- 
stituting the  corona  radiata,  which  still  show  the  radial  arrangement 
about  the  egg,  are  becoming  detached  from  one  another.  As  in  the  other 
two  follicles,  the  first  polar  cell  has  been  produced,  and  the  second 
spindle  (not  shown  in  the  drawing)  is  fully  formed.  There  is  a  small 
space  between  the  zona  pellucida  and  the  vitellus. 

In  fig.  39  (plate  6)  the  contents  of  the  follicle  have  begun  to  flow 
out  into  the  periovarial  space  through  an  opening  at  the  surface  of  the 
ovary.  The  opening  does  not  have  the  appearance  one  would  expect 
to  result  from  a  rupture  due  to  pressure  from  within,  but  rather  from  a 
condition  produced  by  the  migration  of  cells  away  from  the  rupturing 
region.  The  viscidity  of  the  fluid  is  indicated  by  the  sinuous,  more  or 
less  parallel,  line-like  markings  of  the  escaping  contents  (see  also  Sobotta, 
1895) ,  and  the  plasticity  of  the  discus  cells  is  shown  by  the  partial  oblitera- 
tion of  the  radial  arrangement  of  the  corona  cells  around  the  egg.  The 
distance  between  zona  and  vitellus  is  so  much  increased  on  the  deep 
side  of  the  egg  that  the  polar  cell  lies  in  the  space  thus  formed  quite  free 
from  contact  with  either. 

In  the  last  stage  (fig.  40)  the  egg  lies  in  the  periovarial  space,  the 
follicle  having  collapsed.  Here,  too,  there  is  the  same  lack  of  evidence 
of  a  violent  tearing  of  the  follicle  wall.  The  contents  of  the  follicle  still 
have  the  appearance  of  a  viscous  substance.  The  flattening  of  the  egg, 
probably  caused  by  unequal  pressure — perhaps  due  to  the  narrowness 
of  the  space  between  the  ovarian  capsule  and  the  wall  of  the  ovary- 
suggests  considerable  plasticity.  This  condition  can  also  be  seen  sub- 
sequently in  eggs  which  lie  between  ridges  of  the  oviduct.  The  zona  is 
separated  from  the  vitellus,  as  in  the  preceding  stage,  and  the  polar 
cell  is  detached  from  the  egg,  though  not  shown  in  fig.  40. 

The  corona  cells  surround  the  egg  in  its  passage  to  the  oviduct  and 
persist  for  a  varying  number  of  hours. 


24      THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

VI.  SIZE  OF  EGG. 

All  measurements  made  to  determine  the  size  of  eggs  at  different 
stages  of  maturation  have  been  made  on  eggs  fixed  in  the  same  way  and 
measured  with  the  same  objective  and  the  same  eyepiece  and  micro- 
meter. The  diameter  does  not  include  the  zona  pellucida.  Since  the 
egg  is  seldom  quite  spherical  the  longest  and  the  shortest  diameter  of 
the  middle  section  of  the  series  into  which  each  egg  was  cut  was  meas- 
ured. Half  the  sum  of  these  two  measurements  was  taken  as  the  diam- 
eter of  the  egg. 

Table  2  (p.  14)  shows  clearly  the  changes  in  size  of  the  ovum  as 
it  advances  in  maturation.  Under  the  heading  "Diameter  of  eggs" 
the  first  column  gives  the  number  of  eggs  measured;  the  second  column, 
the  average  diameter  of  all  these  eggs;  the  third  and  fourth  columns,  the 
diameters  of  the  largest  and  the  smallest  eggs  of  each  lot  measured. 

It  will  be  seen  that,  with  one  exception,  there  is  a  steady  decrease 
in  size  from  Stage  I  to  Stage  VII.  The  exception,  Stage  II,  shows  only 
a  slight  deviation  and  is  probably  due  to  the  fact  that  the  average  is 
based  on  so  small  a  number  (13)  of  eggs.  Stages  IVa  and  IV6,  hitherto 
treated  by  us  as  Stage  IV,  show  the  same  progressive  decrease.  There 
is  a  small  reduction  in  size  at  the  time  the  first  polar  cell  is  formed 
(Stage  VI),  and  another  in  Stages  VII  and  VIII,  when  the  eggs  have 
left  the  ovary  and  have  been  in  the  oviduct  for  only  a  short  time.  Pos- 
sibly the  fact  that  Stage  VII  is  not  intermediate  in  value  between  Stages 
VI  and  VIII  may  be  due,  as  presumably  in  Stage  II,  to  the  small  num- 
ber (10)  of  eggs  on  which  the  average  is  based.  Eggs  that  were  observed 
in  the  oviduct  about  29  hours  or  more  p.p.  show  a  slight  increase  in 
size  (see  foot-note  to  table  2).  The  sizes  in  the  remaining  stages  can 
have  no  special  meaning  because  the  eggs  had  been  in  the  oviduct  vary- 
ing lengths  of  time. 

Aside  from  the  change  in  volume,  there  is,  as  the  column  of  maxi- 
mum and  minimum  diameters  shows,  considerable  individual  variation. 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  25 

VII.  OBSERVATIONS  ON  THE  MATURATION  PROCESSES. 

In  considering  the  various  topics  of  maturation  the  processes  are 
dealt  with  by  stages,  the  chief  characteristics  of  which  have  been  briefly 
suggested  in  table  2  (p.  14).  It  seems  desirable,  however,  to  give  a  more 
precise  definition  of  these  stages  before  proceeding  to  a  detailed  account 
of  maturation. 

It  should  be  borne  in  mind  that  these  stages,  though  fairly  distinct, 
are,  nevertheless,  only  periods  in  a  continuous  process  of  development 
and  therefore  connected  with  each  other  by  intermediate  conditions. 

A.  OOCYTE  I. 
l.  GENERAL  DESCRIPTION  OF  STAGES. 

STAGE  I. — GERMINATIVE  VESICLE. 

The  germinative  vesicle,  nearly  up  to  the  time  when  it  is  trans- 
formed into  the  first  maturation  spindle,  presents  the  following  condi- 
tions (compare  plate  i,  fig.  i): 

It  is  somewhat  eccentric  in  position,  nearly  spherical,  and  from  19 
to  26  (on  the  average  23)  micra  in  diameter.  It  has  a  uniformly  thin, 
lightly  staining,  smooth  membrane,  and  is  filled  with  a  clear,  homo- 
geneous substance,  the  karyoplasm.  At  one  side  lies  the  vesicular 
nucleolus,  usually  in  contact  with  the  nuclear  membrane.  Immediately 
inside  the  membrane,  and  particularly  around  the  nucleolus  (plasmo- 
some),  are  masses  of  chromatic  substance  attached  to  these  structures 
by  achromatic  material  of  irregular,  though  often  threadlike,  form. 
There  are  a  few  strands,  remnants  of  the  linin  network  of  an  earlier 
stage,  running  through  the  karyoplasm.  Figure  i,  plate  i,  illustrates 
these  conditions,  except  for  the  condition  of  the  nuclear  membrane. 

The  spheroidal,  or  sometimes  lenticular,  nucleolus  is  about  8.5  micra 
in  its  longest  diameter,  and  has  a  fairly  thick,  deeply  staining  wall  of  uni- 
form thickness.  It  contains  only  a  clear,  homogeneous  substance,  never 
any  chromatic  bodies  such  as  are  attached  to  its  outer  surface,  either  as 
distinct  bodies  or  as  apparent  thickenings  of  its  membrane  (fig.  i). 

The  chromatic  masses  of  the  nucleus  are  usually  globular,  though 
sometimes  of  an  irregular  form,  and  have  no  correspondence  with  chro- 
mosomes of  later  stages  either  in  number  or  in  shape.  In  phospho- 
tungstic-acid  haematoxylin  some  of  them  are  stained  deep  blue,  like 
chromatin;  a  few  pink,  like  cytoplasm.  There  are  in  addition  deeply 
stained  granules  scattered  through  the  nucleus.  These  are  usually 
associated  with  the  achromatic  substance. 

Preparatory  to  the  advent  of  the  first  spindle,  the  germinative 
vesicle  moves  a  little  nearer  the  surface  of  the  egg,  but  the  depth  at 
which  it  comes  to  lie  is  not  the  same  in  all  cases.  It  then  decreases  in 
size,  and  its  membrane  becomes  a  little  fainter  and  presents  a  very 
irregular,  wrinkled  appearance  (fig.  i). 


26     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

STAGE  II. — FORMATION  OF  FIRST  MATURATION  SPINDLE. 

The  passage  from  the  preceding  stage  to  this  one  is  rapid.  The 
germinative  vesicle  has  shrunk  still  more  and  is  surrounded  by  a  narrow, 
clearer  region,  in  which,  however,  there  are  cytoplasmic  granules  (figs. 
2  and  3).  Its  contents  are  no  longer  clear  and  homogeneous,  but  show 
a  granular  condition,  much  like  that  of  the  immediately  surrounding 
cytoplasm.  A  few  achromatic  threads  are  still  visible.  The  nucleolus 
and  chromatic  spherules  have  disappeared  (compare  fig.  i  with  figs.  2 
and  3),  and  instead  there  is  a  group  of  chromosome  bodies,  which  is 
usually  located  at  one  side,  rather  than  in  the  middle,  of  the  nucleus. 

Figs.  2,  3,  4,  and  5  show  the  first  steps  in  the  formation  of  the  first 
maturation  spindle.  The  fundaments  of  the  chromosomes  differ  greatly 
in  form.  Some  are  masses  of  irregular  shape,  which  it  is  hard  to  dis- 
tinguish from  the  large  granules ;  some  are  ring-like ;  a  few  are  elongated 
and  show  a  simple  or  a  compound  curve ;  still  others  show  divisions  into 
two  or  four  parts  (figs.  2  and  3).  Later  (figs.  4  and  5),  these  all  become 
completely  differentiated  and  assume  more  definite  and  characteristic 
forms,  some  in  advance  of  others.  As  they  assume  more  precise  forms 
they  become  more  separated  from  one  another.  Their  number  is  at 
first  uncertain,  but  by  the  time  they  have  reached  the  condition  seen 
in  figs.  4  and  5  it  is  clearly  20  (see  table  2,  Stage  II,  p.  14). 

At  an  early  stage  in  their  development  the  fundaments  of  the  chro- 
mosomes lie  in  a  group  at  one  side  of  a  homogeneous  portion  of  the 
karyoplasm  which  is  denser  than  the  surrounding  nuclear  contents 
(figs.  3  and  4).  This  denser  portion,  at  first  indefinite  in  form  (fig.  4), 
increases  in  size  and  develops  into  the  first  maturation  spindle.  As  it 
grows  the  chromosomes  move  apart  and  all  come  to  lie  at  its  surface. 
At  length  it  becomes  elliptical  in  outline  (fig.  5) ,  and  then  shows  delicate 
fibrillations  extending  from  pole  to  pole.  At  the  same  time  the  sub- 
stance of  the  spindle  becomes  less  homogeneous,  showing  granules  dis- 
tributed through  it,  so  that,  except  for  the  fibrillations,  it  becomes  in 
appearance  more  like  the  rest  of  the  karyoplasm.  Meanwhile,  the  clear 
zone  around  the  nuclear  membrane  disappears  (figs.  2  to  5) ,  and  at  the 
same  time  the  general  contents  of  the  germinative  vesicle  assume  more 
nearly  the  appearance  of  the  surrounding  cytoplasm;  the  nuclear  mem- 
brane, which  meanwhile  has  shrunk  little,  if  any,  more,  is  gradually 
dissolved  (fig.  5),  vanishing  more  quickly  in  some  regions  than  in  others. 
Its  disappearance  may  begin  in  some  parts  very  early  (fig.  36). 

STAGES  III  TO  V. — DEVELOPMENT  AND  DIVISION  OF  FIRST  MATURATION  SPINDLE. 
Stage  III  (plate  i,  figs.  6,  7,  and  70). — With  the  complete  disap- 
pearance of  the  membrane  of  the  germinative  vesicle  the  spindle  is 
left  free  in  the  midst  of  the  cytoplasm.  It  is  broadly  elliptical  (fig.  6) 
and  shows,  not  only  on  its  surface  but  in  the  interior  as  well,  very  fine 
fibrillations,  which  conform  in  direction  to  its  shape.  As  in  Stage  II, 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  27 

there  are  granules  scattered  throughout  its  substance  and  the  chromo- 
somes are  still  distributed  over  its  surface.  Sometimes  the  surrounding 
cytoplasm  shows  a  faint  radial  structure,  which  has  the  axis  of  the  spindle 
at  its  center  (fig.  7). 

Stage  IVa  (plate  2,  figs.  8,  8a,  86,  and  9). — In  this  stage  the  chromo- 
somes are  drawn  into  the  region  of  the  equatorial  plane,  some  lying  at 
the  surface  and  some  nearer  the  axis  of  the  spindle,  where  all  make  up 
a  cluster  having  the  form  of  an  uneven  disk,  the  so-cal1.ed  equatorial 
plate.  The  spindle  fibers  are  still  very  delicate.  Occasionally  the  radial 
structure  of  the  surrounding  cytoplasm  seen  in  the  preceding  stage  can 
still  be  observed  (fig.  8). 

Stage  IV6  (plates  2  and  3,  figs.  12  to  14). — The  chromosomes,  still 
near  the  plane  of  the  equator  of  the  spindle,  are  sometimes  visibly  attached 
to  the  spindle  fibers,  which  are  now  much  more  easily  seen.  However,  the 
chief  characteristics  of  this  stage  are  the  tormation  of  several  circum- 
polar  bodies  at  each  end  of  the  spindle  and  the  appearance  of  a  clearer 
cytoplasmic  region  surrounding  the  spindle  on  all  sides.  The  spindle  in 
this  stage  begins  to  elongate  and  to  become  correspondingly  narrower. 

Stage  V  (plates  3  and  4,  figs.  14  to  17). — This  stage  is  characterized 
by  the  division  and  separation  of  the  chromosomes  (metaphase  and  ana- 
phase  of  nuclear  division).  Fig.  14  shows  several  chromosomes  already 
divided  into  halves,  while  others  are  in  process  of  separation.  Figs.  15,16, 
and  1 7  show  more  advanced  stages  in  the  migration  of  the  daughter  chro- 
mosomes toward  the  poles  of  the  spindle  and  also  an  increasing  diminution 
in  the  number  and  size  of  the  circumpolar  bodies  and  in  the  extent  of  the 
clear  region  in  the  neighboring  cytoplasm.  The  more  advanced  represent- 
atives of  this  stage  (figs.  16  and  17)  show  thickenings  of  the  interzonal 
filaments  midway  between  their  ends,  and  also  the  beginning  of  the  con- 
striction which  cuts  off  the  first  polar  cell. 

STAGE  VI. — TELOPHASE  OF  FIRST  SPINDLE  AND  THE  FIRST  POLAR  CELL. 

(PLATE  4,  FIG.  18.) 

In  this  stage  the  daughter  chromosomes,  both  in  the  egg  and  in 
the  polar  cell,  have  fused  into  compact  masses,  which  are  still  joined 
to  each  other  by  the  interzonal  filaments.  The  middle  thickenings  of 
the  filaments  have  united  to  form  the  "cell  plate,"  which  is  continuous 
at  its  edge  with  the  vitelline  membrane  where  the  latter  has  been  con- 
stricted to  form  the  neck  of  the  polar  cell.  The  circumpolar  bodies  have 
disappeared  and  the  clear  cytoplasmic  region  is  very  pale. 

2.  CHROMATIN  PARTS  OF  FIRST  MATURATION  SPINDLE. 

The  origin  of  the  fundaments  of  the  chromosomes  has  already  been 
described  (p.  26).  Although  we  are  unable  to  state  how  these  funda- 
ments are  formed  from  the  chromatin  of  the  germinative  vesicle,  the 
changes  by  which  they  are  converted  into  the  characteristic  mature 
chromosomes  can  be  traced  with  a  fair  degree  of  certainty. 


28     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

Fully  formed  chromosomes  are  shown  in  plate  A,  fig.  G  (e  to  j)  and  in 
figs.  6  to  14  (plates  i  to  3).  In  fig.  G  a  typical  chromosome  is  shown  at  / 
in  face  view  (i.e.,  looking  toward  the  axis  of  the  spindle  along  that 
radius  of  its  equator  which  passes  through  the  middle  of  the  chromo- 
some), and  at  e  in  side  view  (i.e.,  looking  in  the  direction  of  the  tangent 
to  the  equator  of  the  spindle  which  cuts  the  chromosome  at  its  middle 
point) .  To  the  pointed  ends  of  the  chromosome  are  attached  the  spindle 
fibers;  the  side  of  e  (fig.  G)  which  is  directed  to  the  right  is  that  which 
is  turned  away  from  the  axis  of  the  spindle.  The  chromosome  is  com- 
posed of  four  deeply  stained  parts,  which  are  more  or  less  completely 
separated  from  one  another  by  two  deep  constrictions,  one  longitudinal, 
the  other,  less  complete,  transverse.  In  a  sense  the  separation  is  incom- 
plete in  both  directions,  because  the  four  deeply  stained  parts  are  con- 
nected to  one  another  by  a  less  deeply  stained  substance,  in  which  they 
are,  as  it  were,  embedded.  This  substance  may  possibly  be  in  part  non- 
chromatic,  but  probably  it  contains  a  certain  amount  of  chromatin.  This 
diminution  in  the  proportion  of  chromatin  is  also  evident  at  the  pointed 
ends  of  the  chromosomes,  where,  as  already  stated,  the  spindle  fibers  are 
attached  (see  e  and  /).  The  chromosome  illustrated  by  the  two  views 
g  and  h  differs  from  that  seen  in  e  and  /  chiefly  in  being  more  elongated, 
the  four  median,  deeply  stained  regions  of  h  being  the  upturned  adjacent 
ends  of  the  four  parts  resulting  from  the  elongation  of  the  correspond- 
ing thicker  four  parts  shown  in  /.  In  both  these  cases  the  transverse 
division  is  less  conspicuous  than  the  longitudinal.  In  /  both  divisions 
are  obscured  by  the  temporary  fusion  or  adhesion  of  the  four  parts. 
The  cross-division  is,  however,  represented  by  a  constriction.  To  one 
or  other  of  these  three  conditions  can  be  referred  all  the  other  forms  of 
the  fully  developed  chromosomes,  the  differences  being  due  merely  to 
various  degrees  of  fusion  or  separation  of  the  parts.  All  of  these  chro- 
mosomes ultimately  lie  with  their  long  axes  approximately  parallel  to 
that  of  the  spindle. 

We  return  now  to  the  fundaments  of  the  chromosomes  and  their 
development  into  the  forms  last  described.  It  is  to  be  noted  that  in  the 
early  stages  (figs.  2  and  3)  some  fundaments  show  only  a  single  (longi- 
tudinal) division.  The  transverse  division,  seen  clearly  in  the  left-hand 
chromosome  of  fig.  40,  arises  a  little  later,  as  may  be  inferred  from  the 
condition  shown  in  the  lower  right  chromosome  of  fig.  4a  and  in  the 
lower  (pale)  chromosome  of  fig.  5 ;  this  division  may  perhaps  arise  much 
later.  The  4-part  condition  appears  to  be  a  typical  one.  When  it  per- 
sists as  late  as  the  time  of  the  formation  of  the  spindle,  the  chromo- 
some generally  lies  with  its  long  axis  parallel  to  that  of  the  spindle  (fig. 
5).  Were  there  no  forms  intermediate  between  this  and  the  one  shown 
in  /  (fig.  G),  the  four  parts  of  the  one  might  be  referred  in  all  cases  di- 
rectly to  the  corresponding  parts  of  the  other.  But  the  forms  b  and  c 
(d  answering  for  the  face  view  of  both  b  and  c)  are  apparently  intermedi- 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  29 

ate  between  a  and  e,  for  the  stages  of  nuclear  metamorphosis  illustrated 
in  figs.  5,  6,  7,  and  ya,  which  exhibit  these  forms  of  chromosomes  (b  and 
c)  are  in  other  respects  intermediate  between  the  conditions  shown  in 
fig.  4  and  those  of  figs.  8  and  9,  which  present  respectively  the  forms  of 
chromosomes  shown  diagrammatically  in  a  and  /  (plate  A,  fig.  G). 

Owing  to  the  lack  of  exact  synchronism  in  the  formation  of  the 
chromosomes,  it  is  impossible  to  say  with  certainty  which  of  the  forms 
b  and  c  precedes  the  other,  or  even  to  assert  that  they  are  not  independ- 
ent of  each  other.  If  they  do  represent  successive  conditions  of  one  and 
the  same  chromosome,  it  might  be  imagined  that  the  condition  b  had 
been  brought  about  by  a  secondary  union  of  the  four  parts  of  such  a 
chromosome  as  is  shown  in  a,  followed  by  a  bending  in  the  equatorial 
region,  and  that  the  condition  c  was  afterwards  reached  simply  by  a 
thickening  of  the  chromosome  in  the  region  of  the  bending;  but,  on  the 
other  hand,  the  reverse  sequence  might  have  occurred,  and  it  may  be 
urged  in  support  of  this  view  that  c  and  b  represent  respectively  the 
stages  e  and  g,  differing  from  the  latter  chiefly  in  the  obliteration  of  the 
cross-division,  the  one  corresponding  with  the  equator  of  the  spindle. 
As  the  sequence  e  g  seems  the  more  natural  one  for  those  two  forms, 
so  in  the  former  the  sequence  c  b  would  be  a  natural  inference.  The 
basis  for  the  conclusion  that  the  forms  b  and  c  pass  through  a  stage  cor- 
responding to  a  is  the  apparent  absence  of  those  forms  (b  and  c)  in  the 
earlier  stages  of  nuclear  metamorphosis  and  the  prevalence  of  the  a  con- 
dition. It  must,  however,  be  borne  in  mind  that  this  does  not  amount 
to  a  demonstration,  and  that  individual  variations  in  eggs  or  slight  differ- 
ences in  preservation  may  afford  the  real  explanation  of  the  conditions. 

In  b  and  c  the  transverse  division  of  the  earlier  stage,  a,  has,  then, 
either  vanished  by  fusion,  or  has  not  yet  appeared,  whereas  the  longi- 
tudinal one  is  quite  evident  (plate  A,  fig.  G,  d,  and  plate  i,  figs.  7  and  7a). 
At  the  ends  of  the  chromosome, where  the  spindle  fibers  are  attached  (d), 
the  chromatin  is  less  deeply  stained,  as  also  in  /.  The  change  from  the 
condition  seen  in  d  to  that  of  /  is  accomplished  either  by  the  reappearance 
of  the  transverse  division,  or,  in  case  it  had  not  existed  in  the  fundament, 
by  the  first  appearance  of  a  cross-division.  There  is  no  reason,  however, 
to  suppose  that  the  form  /  might  not  in  some  cases  arise  directly  from  a, 
the  transverse  division  never  being  obscured.  As  figs.  4,  5,  and  6  (plate  i) 
show,  some  chromosomes  develop  more  rapidly  than  others. 

The  individual  chromosomes  differ  somewhat  in  size  and  all  seem 
to  become  a  little  smaller  as  they  approach  completion.  They  are  at 
first  distributed  over  the  surface  of  the  spindle  only.  After  they  have 
become  concentrated  in  the  region  of  the  equatorial  plane,  some  are 
still  found  at  the  surface,  but  others  are  in  the  interior  of  the  spindle. 
Even  at  the  beginning  of  metakinesis  all  do  not  lie  exactly  in  the 
equatorial  plane  (fig.  136).  For  this  reason  in  cross-sections  of  spindles 
many  of  the  chromosomes  are  cut  in  two;  polar  views  of  the  "equatorial 
3 


30     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

plate  "are  therefore  unsatisfactory  for  counting  chromosomes.  It  is 
an  interesting  fact  that  in  the  spindles  drawn  in  figs.  10  and  n  (plate 
2)  the  chromosomes  lie  nearer  that  end  of  the  spindle  which  is  more 
pointed  and  about  which  the  evidences  of  cytoplasmic  radiations  are 
more  pronounced. 

The  chromosomes  are  oriented  with  their  long  axes  parallel  to  the 
long  axis  of  the  spindle.  The  few  exceptions  may  in  some  instances  be 
natural,  but  in  others  they  -certainly  are  due  to  displacement  by  the 
knife  in  cutting  (figs.  12,  136,  x  and  #'). 

The  separation  to  form  the  daughter  chromosomes  always  takes 
place  at  the  middle  of  the  chromosome  and  at  right  angles  to  its  long  axis 
(plate  A,  fig.  G,  f  to  /).  While,  in  general,  all  the  daughter  chromosomes 
migrate  toward  the  spindle  poles  at  the  same  time  (fig.  15),  it  sometimes 
happens  that  one  or  more  of  the  chromosomes  divides  and  the  halves 
move  apart  at  an  early  stage  before  their  sister  chromosomes  show  any 
signs  of  migration  (two  pairs  in  fig.  9) .  In  the  latter  case  the  precocious 
daughter  chromosomes  show  no  longitudinal  division,  while  in  the  former 
they  are  clearly  split  lengthwise  (plate  A,  fig.  G,  i,  I;  plate  3,  fig.  15). 
Fig.  1 5  shows  a  spindle  which  is  nearly  parallel  to  the  surface  of  the  egg ; 
in  this  case  each  daughter  chromosome  consists  of  halves,  each  of  which 
is  elongated  and  somewhat  tapering,  the  narrower  end  being  directed 
toward  the  pole  of  the  spindle;  the  halves  are  parallel  to  each  other  or 
slightly  converging  toward  the  ends  which  point  to  the  pole.  In  another 
spindle,  of  like  age  but  occupying  a  radial  position  in  the  egg,  the  halves 
of  each  daughter  chromosome  are  in  contact  at  their  polar  ends,  but 
widely  separated  at  the  equatorial  end,  thus  together  forming  a  distinct 
V.  In  fig.  17  the  daughter  chromosomes  are  more  compact,  and  fewer 
show  the  longitudinal  division.  Some  of  them  are  much  more  elongated 
than  others.  The  spindle  in  plate  3 ,  fig.  1 6 ,  being  cut  obliquely,  shows  the 
daughter  chromosomes  more  clearly.  The  two  limbs  of  each  daughter 
chromosome  are  easily  distinguishable,  each  being  somewhat  dumb-bell 
shaped.  The  two  lie  side  by  side,  and  in  some  cases  by  bending  assume 
the  form  of  flattened  rings  (fig.  166).  Later  the  chromosomes  at  each 
end  of  the  spindle  fuse  into  a  compact,  deeply  staining,  disk-shaped,  or 
sometimes  cup-like,  mass  (plate  4,  fig.  18). 

In  spite  of  the  differences  of  opinion  which  have  been  expressed 
concerning  the  number  of  chromosomes,  we  think  there  can  be  no  doubt 
that  typically  in  the  animals  we  have  studied  it  is  20.  A  knowledge  of 
the  structure  of  the  chromosomes  makes  it  possible  in  many  cases  to 
be  absolutely  sure  that  this  is  the  number.  Table  2  gives  the  results  of 
our  observations  on  this  subject.  The  accuracy  of  the  counting  depends 
on  the  stage  of  the  spindle  and  the  position  which  it  occupies  with  respect 
to  the  plane  of  cutting.  When  the  chromosomes  are  scattered  along  the 
spindle  (figs.  6,  7,  and  7 a),  they  obscure  one  another  least  and  frequently 
can  be  counted  with  perfect  accuracy.  Upon  the  formation  of  the 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  31 

"equatorial  plate,"  however,  they  become  crowded,  and  the  crowding 
increases  as  division  approaches.  Figs.  15  and  16  illustrate  exception- 
ally favorable  cases,  in  which  the  number  can  be  determined  satisfac- 
torily, at  least  at  one  end  of  the  spindle.  It  rarely  happens  that  a  spindle 
lies  wholly  in  one  section;  it  is  usually  cut  into  two  or  three  parts.  This 
is  frequently  of  advantage.  (See  figs.  7  and  70,  8a  and  Sb,  loa  and  lob, 
etc.)  When  the  axis  of  the  spindle  is  parallel  to  the  plane  of  cutting, 
the  chromosomes,  which  are  hardened  by  the  process  of  preservation, 
are  seldom  cut  by  the  knife,  but  are  pushed  to  one  side.  Sometimes 
they  are  dragged  out  of  place  (figs.  i2,x,  136,  x  and  x') ,  or  even  completely 
out  of  the  spindle  into  the  cytoplasm  (fig.  12),  where  they  lie  at  the 
surface  of  the  section  on  the  side  of  the  spindle  toward  which  the 
knife  moved.  In  the  spindle  shown  in  fig.  12  the  chromosomes  (not  all 
of  which  are  drawn)  number  20,  including  the  one  lying  to  the  left  of 
the  spindle.  This  fact,  the  displacement  of  chromosomes,  doubtless 
accounts  for  some  of  the  cases  where  there  seem  to  be  fewer  than  20. 
In  the  spindle  shown  in  figs.  130,  and  136,  for  example,  where  there  are 
only  1 8,  displacement  is  clearly  shown  in  two  chromosomes  (x  and  #') 
lying  at  the  upper  surface  of  the  lower  section  (136);  and  it  is  quite 
possible  that  others  have  been  completely  removed. 

3.  ACHROMATIN  PARTS  OF  FIRST  MATURATION  SPINDLE. 

The  origin  of  the  spindle  has  been  described  under  Stage  II.  At 
first  broadly  elliptical,  it  changes  its  form,  becoming  slightly  sharper 
at  the  poles  and,  on  the  average,  longer  and  narrower,  especially  in  the 
later  stages,  as  division  approaches.  The  fibers  are  not  limited  to  the 
surface  of  the  spindle,  nor  to  any  part  of  it,  but  are  uniformly  distrib- 
uted, as  can  be  seen  in  cross-sections  of  the  spindle.  They  do  not  con- 
verge as  straight  lines  to  a  point,  but  curve  inward  toward  the  poles, 
without,  however,  meeting  (figs.  8,  9,  n  left  end,  12,  i$a,  136,  140). 
Consequently  they  are  never  parallel,  and  the  spindle  poles  are  more 
or  less  open.  However,  in  two  otherwise  apparently  normal  spindles 
(figs.  10,  n)  the  fibers  at  one  pole  do  meet  at  a  point,  from  which  there 
are  a  few  radiations  extending  into  the  surrounding  cytoplasm. 

Besides  the  change  in  proportions,  there  is  also,  on  the  average,  a 
small  increase  in  volume.  At  Stages  III,  IVa,  and  IV6  the  average 
dimensions  are,  respectively,  in  micra,  18.7  X  10.4,  19.2  X  10.8,  and 
22.4  X  9.9.  The  variations  in  size  in  each  stage  are  considerable  (see 
table  2,  p.  14).  With  metakinesis  the  spindles  elongate  considerably 
and  become  narrower.  Three  such  spindles,  parallel  or  nearly  parallel 
to  the  surface  of  the  egg  (fig.  15),  give  as  an  average  a  length  of  26  micra 
and  a  diameter  of  8  micra;  another,  almost  exactly  radial  in  position, 
gives  the  corresponding  measurements  of  23  X  n  micra. 

As  the  spindle  develops,  the  fibers,  at  first  in  the  young  spindle 
evident  only  as  feeble  fibrillations,  become  more  distinct.  They  are 


32     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

usually  smooth  in  appearance  and  of  uniform  size  from  end  to  end  (figs. 
8a,  86,  9,  10,  n).  A  little  later  they  often  exhibit  minute,  granular 
thickenings  at  irregular  intervals  along  their  lengths  (figs,  n,  i$a,  136, 
140).  The  polar  ends  of  the  fibers  become  thickened  and  more  or  less 
confluent  in  the  later  stages  (IV6  and  V;  figs.  12,  130,,  136,  14,  15), 
frequently  to  such  an  extent  that  the  end  of  the  spindle  looks  homo- 
geneous, and  the  fibers  are  distinguishable  only  as  faint  striations  (fig. 
15).  In  some  cases  the  attachment  of  some  of  the  fibers  to  chromosomes 
is  evident  (figs.  10,  n,  i$a,  136,  14,  140).  In  addition  to  these  fibers 
there  are,  however,  others,  very  delicate  ones,  running  from  pole  to  pole 
without  being  attached  to  any  chromosome  (figs.  i$a,  136).  These 
probably  constitute  a  part  of  the  interzonal  filaments.  The  latter,  when 
the  daughter  chromosomes  have  separated,  are  very  fine  (fig.  15).  Later 
(figs.  1 6  and  17)  they  become  thicker,  and  in  the  telophase  (fig.  18)  they 
apparently  become  fused  into  a  pale,  nearly  homogeneous,  faintly  stri- 
ated bundle,  lying  between  the  two  deeply  stained  masses  resulting 
from  the  confluence  of  the  chromosomes.  The  chromosomes,  drawn 
nearly  to  the  end  of  the  spindle,  lie  in  a  somewhat  deeply  staining  matrix 
(fig.  17),  which  is  perhaps  derived  from  the  spindle  fibers. 

At  the  middle  of  each  interzonal  filament  is  a  thickening,  a  "Zwi- 
schenkorperchen. ' '  The  number  of  these  was  not  determined.  The  thick- 
enings, at  first  elongated,  become  more  globular  (fig.  17),  and  at  length  by 
fusion  give  rise  to  the  "cell  plate"  (fig.  18),  a  disk-shaped  mass  staining 
moderately  deeply.  The  further  fate  of  the  interzonal  filaments  and  the 
"Zwischenkorperchen"  will  be  discussed  later  (pp.  34  and  43). 

4.  CENTROSOMES,  CIRCUMPOLAR  BODIES,  AND  CLEAR  REGION. 

Although  recently  the  existence  of  centrosomes  in  connection  with 
the  first  maturation  spindle  in  the  ovum  of  the  mouse  has  been  asserted, 
the  evidence,  so  far  as  our  preparations  show,  points  clearly  to  the  entire 
absence  of  centrosomes.  Not  even  in  the  two  cases  illustrated  in  plate  2, 
figs.  10,  n,  is  there  any  hint  of  a  centrosome  at  the  ends  where  the  fibers 
converge  to  a  point,  although  there  are  clearly  a  few  fiber-like  radiations 
in  the  surrounding  cytoplasm.  If  there  were  any  centrosomes  present, 
one  would  expect  them  to  stain  as  sharply  as  those  in  the  surrounding 
follicle  cells  during  division.  In  the  eggs  from  which  figs.  10  and  n  were 
drawn  there  are  no  polar  radiations  except  those  figured  and  mentioned 
above,  nor  have  any  other  instances  been  observed  in  which  there  were 
polar  radiations  as  marked  as  these.  Occasionally  a  few  fibers  may  be 
observed  outside  the  limits  of  the  spindle  (figs.  9,  11,  12,  136)  and  ex- 
tending from  the  poles  obliquely  toward  the  plane  of  the  equator. 

The  two  conditions  mentioned  as  characteristic  of  Stage  IV6  are 
the  circumpolar  bodies  and  the  clear  region  around  the  whole  spindle. 
The  two  arise  at  about  the  same  time  and  likewise  disappear  together; 
they  both  reach  their  greatest  prominence  at  the  stage  when  the  chro- 
mosomes divide — at  metakinesis. 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  33 

The  circtimpolar  bodies  have  been  so  named  because  they  are 
grouped  around  the  poles  of  the  spindle  (figs.  13,  14).  Their  origin  is 
not  known  beyond  the  fact  that  they  come  into  existence  gradually  at 
the  spindle  poles.  They  are  variously  shaped  (figs.  i$a,  136,  14,  i^a, 
15),  no  one  form  having  predominance  over  others.  Some  have  irreg- 
ular forms  or  are  roughly  spherical,  others  are  pear-shaped,  still  others 
disk-like.  In  ordinary  plasma  stains  they  are  very  inconspicuous, 
apparently  being  composed  of  a  homogeneous  substance  somewhat 
denser  than  the  surrounding  cytoplasm.  In  phosphotungstic-acid  hsema- 
toxylin,  on  the  contrary,  they  become  deep  blue,  like  the  chromosomes, 
from  which  they  are  distinguishable  only  by  their  forms.  They  appar- 
ently have  no  connection  with  the  spindle  fibers  (figs.  13*2,  136,  140), 
and  after  the  chromosomes  have  reached  the  ends  of  the  spindle  they 
fade  away  (plate  3,  fig.  16)  and  disappear  altogether  (plate  4,  fig.  18). 

The  clear  region  around  the  spindle  is  often  visible  in  sections  as  a 
faint,  broad  zone  before  the  circumpolar  bodies  appear  (figs,  n  and  12), 
and  it  often  persists  for  a  short  time  after  they  have  vanished  (figs.  16 
and  1 8).  When  most  conspicuous  it  is  comparatively  narrow.  It  ap- 
pears more  homogeneous  than  the  surrounding  cytoplasm  by  reason  of 
its  being  less  granular;  but  at  no  time  is  it  quite  free  from  granules. 

5.  POSITION  AND  ORIENTATION  OF  FIRST  MATURATION  SPINDLE. 

The  depth  at  which  the  spindles  lie  is  variable.  Whether  the  fully 
formed  spindle  remains  at  first  in  the  position  which  was  occupied  by  the 
germinative  vesicle  when  its  membrane  vanished  is  undecided.  At  all 
events,  before  the  time  when  the  chromosomes  divide,  the  spindles  may 
be  found  at  different  depths.  When  the  polar  cell  is  about  to  be  cut  off 
the  spindle  comes  to  lie  near  the  surface  of  the  egg,  assumably  in  the 
region  of  the  animal  pole.  The  axis  of  the  spindle  may  be  parallel, 
oblique,  or  perpendicular  to  a  tangent  to  the  surface  of  the  egg  at  the 
point  nearest  the  spindle.  These  positions  are  not  characteristic  of 
particular  stages,  but  may  be  found  at  any  epoch  in  the  maturation. 
The  perpendicular  position  is  least  often  met  with,  the  oblique  at  vari- 
ous angles,  and  the  parallel  positions  are  the  most  frequent.  It  seems 
quite  possible  that  the  spindle  maintains  its  original  orientation  when 
it  approaches  the  surface  to  divide.  At  least,  it  is  certainly  true  that 
the  perpendicular  position  is  not  requisite  for  the  formation  of  the  polar 
cell  (see  p.  34),  for  of  ten  spindles  in  the  stages  shown  in  figs.  15,  16,  and 
17,  only  one  was  perpendicular,  the  others  being  either  parallel  or  some- 
what oblique.  The  perpendicular  one  was  in  a  stage  corresponding  to 
that  illustrated  by  fig.  15.  In  nearly  all  examples  of  the  stage  shown  in 
fig.  1 8  the  bundle  of  interzonal  filaments  is  oblique  to  the  radius  of  the 
egg,  though  sometimes  it  varies  only  a  little  from  that  position.  In 
other  cases  it  is  very  much  bent,  apparently  as  a  result  of  a  more  rapid 
ingrowth  of  the  cell  wall  on  one  side  during  abstriction  of  the  polar  cell. 


34     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

6.  ABSTRICTION  OF  FIRST  POLAR  CELL. 

The  process  of  abstriction  begins  as  soon  as  the  daughter  chromo- 
somes have  come  close  to  the  poles  of  the  spindle  and  the  "  Zwischen- 
korperchen"  have  attained  the  condition  shown  in  fig.  17.  While  the 
spindle  may  sometimes  be  perpendicular  to  the  surface  of  the  egg,  as 
already  stated,  one  pole  lying  in  an  elevation  or  protrusion,  the  condi- 
tions indicate  that,  in  most  cases  at 
least,  the  spindle  is  either  parallel  or 
oblique  to  the  surface  (figs.  15,  16,17). 
The  pole  nearer  the  surface  does  not 
at  first  lie  in  the  middle  of  the  protru- 
sion, but  at  one  edge  of  it  (fig.  17). 


•M&  J         The   constricting   process   begins   on 
— '  the  side  nearest   the    "  Zwischenkor- 


perchen,"  where  in  the  surface  of  the 
FIG.  H.  ,    . 

(Compare  figs.  i6a  to  i6d,  plate  3.)       e^  a  deeP>  sharP  groove  brings  the 

vitelline  membrane  into  contact  with 

the  "  Zwischenkorperchen  "  of  the  side  of  the  spindle  nearest  the  surface. 
The  same  condition  exists  also  in  fig.  1 6 ,  in  which  the  plane  of  sectioning 
is  very  oblique  to  the  axis  of  the  spindle,  as  may  be  seen  by  comparison 
with  fig.  H,  which  is  a  diagrammatic  reconstruction  of  an  imaginary 
section  of  the  egg  in  a  plane  perpendicular  to  that  of  the  actual  sections, 
but  parallel  to  the  axis  of  the  spindle.  (Compare  plate  3,  figs.  i6a 
to  i6d.) 

No  other  stage  between  this  and  that  shown  in  fig.  18  having  been 
found,  the  further  steps  in  the  process  can  only  be  inferred.  However, 
it  is  highly  probable  that  the  contact  between  the  vitelline  membrane 
and  the  "Zwischenkorperchen,"  shown  in  fig.  17,  advances  until  it  has 
quite  encircled  the  spindle.  The  result  is  that  the  entire  periphery  of 
a  disk-like  body  formed  by  the  fusion  of  the  "Zwischenkorperchen" 
is  finally  in  contact  with  the  vitelline  membrane  (fig.  18),  and  the  orig- 
inal protrusion,  now  become  more  voluminous  and  containing  the  super- 
ficial group  of  chromosomes,  is  thus  separated  from  the  egg.  The  inter- 
zonal filaments,  brought  into  a  more  nearly  radial  position  during  the 
constriction,  form  the  bulk  of  the  neck  of  the  polar  cell.  A  little  later 
the  constriction  is  completed  by  the  ingrowth  of  the  cell  membranes  of 
both  egg  and  polar  cell  in  such  a  way  as  to  cut  off  the  interzonal  fila- 
ments and  leave  the  "Zwischenkorperchen"  on  the  outside  of  the  cell 
membranes  of  both  polar  cell  and  egg.  Thus  is  formed  the  first  polar 
cell  and  the  oocyte  of  the  second  order. 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  35 

B.  OOCYTE  II. 
1.  GENERAL  DESCRIPTION  OF  STAGES. 

The  chief  criterion  according  to  which  an  egg  may  be  judged  to 
be  an  oocyte  of  the  first  order  or  of  the  second  order  is  the  character  of 
the  chromatin  contents.  As  the  sequel  will  show,  this  is  the  only  relia- 
ble standard.  It  will  naturally  occur  to  the  reader  that  the  oocyte  of 
the  second  order  must  be  accompanied  by  the  first  polar  cell,  and  that 
this  fact  would  be  a  satisfactory  criterion.  But  the  following  facts  com- 
plicate the  situation:  first,  some  fertilized  eggs  exhibit  two  polar  cells, 
some  but  a  single  one;  secondly,  there  is  dispute  as  to  whether  this 
single  polar  cell  is  homologous  with  the  first  or  second  one  of  eggs  hav- 
ing two.  In  the  description  of  the  following  stages  it  will  be  assumed 
that  the  egg  naturally  has  two  polar  cells,  and  the  question  as  to  how 
many  polar  cells  are  actually  formed  will  be  treated  of  in  a  later  chapter. 

STAGE  VII. — FORMATION  OF  SECOND  MATURATION  SPINDLE. 

(PLATE  4,  FIG.  19.) 

It  is  fair  to  infer  from  the  comparatively  long  duration  of  the  pre- 
ceding Stage  (VI)  that  the  disk-shaped  mass  of  chromatin  which  re- 
sults from  the  more  or  less  complete  fusion  of  the  chromosomes  left 
in  the  egg  after  the  formation  of  the  first  polar  cell  probably  remains 
for  some  time  without  perceptible  change  of  morphological  conditions, 
and  that  the  persisting  half  of  the  interzonal  filaments  likewise  under- 
goes little  change  during  this  period.  With  the  close  of  this  period  of 
apparent  inactivity  Stage  VII  begins.  It  embraces  only  the  metamor- 
phosis of  the  chromatin  mass  and  what  are  probably  the  achromatic 
remnants  of  the  first  spindle  into  the  fully  formed  second  maturation 
spindle.  This  process,  unlike  the  one  involved  in  the  completion  of 
the  first  spindle,  is  so  rapid  that  it  can  not  be  subdivided  into  stages 
and  traced  step  by  step. 

STAGE  VIII. — "EQUATORIAL  PLATE"  OF  SECOND  MATURATION  SPINDLE. 

(PLATES  4,  5,  FIGS.  20  TO  27.) 

As  this  stage  is  unique,  in  that  it  depends  on  the  occurrence  of 
semination  for  its  normal  termination,  it  may  have  a  greater  length 
than  any  other  part  of  the  whole  maturation  process,  and  is  therefore 
the  one  most  easily  obtained.  If  semination  is  early,  the  spindle  divides 
without  undergoing  any  previous  alterations;  on  the  other  hand,  if  the 
access  of  spermatozoa  be  hindered,  the  spindle,  though  remaining  com- 
paratively inactive,  undergoes  certain  changes  as  a  result. 

When  newly  formed,  the  second  maturation  spindle  (plates  4  and 
5,  figs.  22  to  24)  is  very  similar  to  the  first  spindle  immediately  before 
its  metakinesis,  differing  from  it  only  in  being  a  little  smaller,  in  the 
structure  of  its  chromosomes,  and  in  their  more  exact  arrangement  in 
the  plane  of  the  equator.  If  semination  is  prevented,  the  resulting  pro- 
longed quiescence  of  the  spindle  is  characterized  by  a  diminution  in 


36     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE.    1 

the  number  of  the  circumpolar  bodies,  and  often  by  their  complete 
disappearance,  and  by  the  disappearance  of  the  clear  region  previously 
described  as  surrounding  the  first  spindle. 

STAGE  IX. — DIVISION  OF  SECOND  MATURATION  SPINDLE. 

(PLATE  5,  FIGS.  28  TO  30.) 

The  separation  of  the  daughter  chromosomes  takes  place,  as  a  rule, 
only  after  a  spermatozoon  has  touched  or  penetrated  the  egg.  How- 
ever, in  the  case  of  one  animal — a  mouse  which  had  not  been  insemi- 
nated— one  of  the  eggs  contained  the  divided  chromosomes  arranged 
in  two  parallel  daughter  plates,  which  were  still  near  the  equator  of  the 
spindle ;  another  egg  from  the  same  mouse  presented  a  stage  still  further 
advanced  (plate  5,  fig.  28),  the  two  groups  of  daughter  chromosomes 
in  this  case  having  migrated  nearer  to  the  poles  of  the  spindle. 

STAGE  X. — TELOPHASE  OF  SECOND  SPINDLE  AND  SECOND  POLAR  CELL. 
(PLATE  5,  FIG.  30.) 

The  beginning  of  the  abstriction  of  the  second  polar  cell  resembles 
that  of  the  first.  This  stage,  indeed,  agrees  so  closely  with  the  corre- 
sponding stage  in  the  formation  of  the  first  polar  cell  (Stage  VI,  p.  27), 
from  which  it  seems  to  differ  only  in  the  presence  in  the  oocyte  of  the 
head  of  a  spermatozoon,  that  it  need  not  be  described  here.  It  may  be 
said  that,  of  30  eggs  in  this  stage,  only  i  failed  to  show  the  head  of  a 
spermatozoon. 

2.  CHROMATIN  PARTS  OF  SECOND  MATURATION  SPINDLE. 

The  chromosomes  of  the  second  maturation  spindle  arise  directly 
from  the  chromatin  mass  which  remains  in  the  egg  after  the  abstric- 
tion of  the  first  polar  cell,  i.e.,  without  an  intervening  vesicular  stage 
of  the  nucleus.  This  mass  breaks  up  into  fragments,  but  whether  or 
not  each  of  these  fragments  is  the  equivalent  of  a  chromosome,  either 
single  or  multiple,  it  is  difficult  to  determine.  Whatever  their  mode  of 
origin,  the  fragments  are  fairly  (or  even  very)  irregular  in  form,  incom- 
pletely separated  from  one  another,  and  of  uncertain  number  (plate  4, 
fig.  19).  Some  of  them  bear  a  slight  resemblance  to  the  daughter  chro- 
mosomes of  the  previous  division  which  had  nearly  reached  the  poles  of 
the  first  spindle  (fig.  16).  Sometimes  (fig.  196)  they  are  embedded  in  a  ma- 
trix of  homogeneous  substance  denser  than  the  surrounding  cytoplasm. 
They  are  never  scattered,  and  soon  become  arranged  in  the  plane  of  the 
equator  of  the  future  spindle,  where  they  may  constitute  a  group  having 
the  form  of  an  imperfect  ring.  No  stages  between  this  and  that  of  the 
completely  formed  chromosomes  have  been  observed. 

The  chromosomes  of  the  completed  second  spindle  (figs.  23  and  24) 
often  closely  resemble  the  daughter  chromosomes  of  the  first  spindle 
as  they  appear  when  they  have  nearly  finished  their  poleward  migra- 
tion (fig.  1 6),  for  each  mother  chromosome  of  the  second  spindle  is  com- 
posed of  a  pair  of  elements,  and  these  elements  vary  in  form,  independ- 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  37 

ence,  and  relative  position  (figs.  23  to  27,  plates  4,  5;  fig.  7).  Fig.  7 
(m  to  r)  illustrates  several  of  these  variations.  In  the  simplest  form  of 
chromosomes,  shown  at  m,  each  element  is  a  straight  rod,  either  of  uni- 
form size  (see  also  fig.  25),  or  slightly  constricted  in  the  middle.  The 
constriction  is  likewise  evident  in  chromosomes  seen  when  looking 
nearly  in  the  direction  of  the  axis  of  the  spindle  (figs.  20,  21).  Viewed 
under  these  conditions  usually  one  element  of  the  pair  is  partially  cov- 
ered by  the  other.  Even  after  the  separation  of  the  daughter  chromo- 
somes from  each  other,  this  constriction  or  dumb-bell  condition  of  the 
daughter  chromosome  is  evident,  whether  seen  endwise  (fig.  28)  or  in 
side  view.  In  p  (fig.  7)  the  constriction  is  carried  still  further,  some- 
times to  such  an  extent  that  the  mother  chromosome  appears  to  be  com- 
posed of  four  nearly  independent  parts  (x,  fig.  240).  Sometimes  the 
daughter  chromosomes  are  curved  rods  (fig.  7,  r;  plate  5,  figs.  246,  26), 
or  are  of  an  irregular  crescent  shape  (o) .  Fusion  or  adhesion  of  the  two 
elements  at  one  or  more  points  gives  rise  to  figures  like  n  (see  also  figs. 
20,  23#,  x).  When  the  elements  are  more  elongated  and  curved,  rings 
(q,  also  figs.  24a,  25,  26)  are  formed  by  the  fusion  of  the  corresponding 
ends  of  the  two  daughter  chromosomes.  Occasionally,  when  the  fusion 
of  the  ends  (as  in  n)  is  well  advanced  and  the  constriction  in  the  middle 
of  each  is  complete,  the  original 
separation  between  the  two  ele- 
ments is  obscured  and  the  mother 
chromosome  then  appears  to  be 
composed  of  two  parts,  the  long  m  n  °  p  q  r 
axes  of  which  are  perpendicular  to  FIG.  7. 

the  plane  of  the  equator.     Forms 

like  those  shown  in  figs.  25  and  2 7, which  occur  in  eggs  that  have  remained 
long  in  the  oviduct,  are  explained  by  the  fact  that  with  age  the  elements 
tend  to  elongate.  In  any  of  these  forms  of  chromosome  the  parts  may 
be  parallel  to  each  other,  or,  according  to  the  point  at  which  the  spindle 
fibers  are  attached,  separated  at  one  end  (n,  p,  r)  or  at  the  middle  (o,  q). 

The  chromosomes  are  never  arranged  at  the  surface  of  the  spindle, 
but  from  the  beginning  are  uniformly  distributed  in  the  plane  of  its 
equator  (figs.  20  to  24),  and  are  so  oriented  that  that  plane  passes  be- 
tween the  two  elements  of  each  mother  chromosome.  This  arrangement 
of  the  daughter  chromosomes  in  one  plane  is  preserved  even  after  meta- 
kinesis  (figs.  280  and  286). 

The  number  of  chromosomes  is  20;  but  the  proportion  of  cases  in 
which  the  number  can  be  determined  with  accuracy  is  smaller  than  in 
the  case  of  the  first  spindle,  because  in  the  second  spindle  the  chromo- 
somes are  more  crowded  and  their  forms  are  less  regular  than  in  the 
first  spindle.  When,  in  cutting,  the  chromosomes  fall  in  two  sections 
the  difficulty  of  counting  is  usually  increased.  However,  knowing  the 
structure  of  the  chromosomes,  it  has  been  possible  in  many  cases  to  be 


38     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

quite  certain  that  the  number  is  20  (see  table  2,  p.  14).  In  figs.  230,  and 
236  there  are  only  19  chromosomes,  one  probably  having  been  lost  in 
cutting.  Figs.  2^0,  and  246  exhibit  together  20,  one  having  been  so  cut 
that  a  half  of  it  lies  in  each  section.  The  two  sections  (24*1  and  246) 
contain,  respectively,  8.5  and  11.5  chromosomes.  Polar  views  of  the 
"equatorial  plate"  are  usually  the  most  satisfactory  ones  for  counting. 
In  fig.  20,  a  polar  view,  there  are  clearly  20  chromosomes;  one  of  these 
(#),  seen  in  face  view,  corresponds  to  fig.  7,  n.  In  figs.  28a  and  286  (an 
anaphase)  the  number  can  not  be  determined  with  perfect  accuracy, 
because  the  long  axes  of  the  daughter  chromosomes  are  perpendicular 
to  the  plane  of  the  section.  Two  of  the  larger  chromosomes  (x  and  x') 
may  well  be  double;  if  so,  the  number  in  this  case  also  is  20. 

In  the  division  of  the  chromosomes,  the  two  elements  of  each 
mother  chromosome  separate  and  then  migrate  to  the  opposite  poles 
of  the  spindle.  Figs.  28a  and  286  (plate  5)  are  polar  views  of  the  two 
daughter  plates  at  a  stage  of  migration  corresponding  to  that  of  fig.  16, 
and  are  drawn  from  a  non-seminated  egg.  In  fig.  29,  which  represents 
a  slightly  later  stage  than  fig.  28,  the  individual  chromosomes  are  no 
longer  distinguishable.  They  seem  quickly  to  lose  their  identity  and 
merge  into  a  single  disk-shaped  mass  (fig.  30),  as  in  the  case  of  the  first 
spindle. 

3.  ACHROMATIN  PARTS  OF  SECOND  MATURATION  SPINDLE. 

The  interzonal  filaments  left  in  the  egg  after  the  first  polar  cell  is 
cut  off  persist  for  a  while  along  with  the  chromatin  mass.  About  the 
time  when  the  chromatin  breaks  up  into  fragments,  they  lose  their  con- 
nection with  the  cell  plate  (plate  4,  fig.  196).  It  is  probable,  but  not  cer- 
tain, that  they  contribute  to  the  formation  of  the  matrix  in  which  the 
chromatin  fragments  are  embedded,  and  also  to  the  formation  of  the 
fibers  of  the  completed  second  maturation  spindle. 

The  second  spindle  begins  as  a  somewhat  pear-shaped,  apparently 
homogeneous  body  at  the  time  when  the  chromatin  mass  divides  into 
fragments.  When  completed  it  is  more  or  less  elliptical  (fig.  22),  like 
the  first  spindle,  but  it  varies  more  in  form  than  does  the  first  spindle, 
being  occasionally  more  slender  and  having  more  sharply  pointed  ends. 
However,  as  observed  from  the  surface  of  the  egg,  it  often  appears  very 
broad  (figs.  23,  24),  owing  to  its  being  flattened  in  the  direction  of  the 
radius  of  the  egg  (fig.  20).  Such  spindles  when  seen  edgewise  appear 
very  narrow  (fig.  22) ;  they  always  lie  nearer  the  surface  of  the  egg  than 
those  which  are  circular  in  cross-section. 

The  fibers  of  recently  formed  spindles  resemble  quite  closely  those 
of  the  later  stages  of  the  first  spindle  in  being  smooth,  of  uniform 
diameter — except  at  their  polar  ends,  where  they  are  thickened — and 
curved  inward  toward  the  poles  (figs.  22  and  24).  The  thickenings  at 
the  polar  ends  are  not  to  be  seen  in  fig.  23,  because  the  spindle  was  stained 
in  Bohmer's  haematoxylin  and  Congo  red,  which  are  not  favorable  for 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  39 

demonstrating  the  fibers  and  circumpolar  bodies  clearly.  As  a  rule, 
the  individual  fibers  and  their  attachment  to  the  chromosomes  are  not 
easily  distinguishable.  Although  one  can  not  be  absolutely  certain  that 
there  are  fibers  which  are  continuous  from  pole  to  pole  without  being 
connected  to  any  of  the  chromosomes,  it  is  perhaps  reasonable  to  assume 
that  such  is  the  case,  because  of  the  general  similarity  of  the  second 
spindle  to  the  first  one,  where  such  a  condition  is  fairly  evident.  The 
daughter  chromosomes,  after  their  migration  toward  the  poles  of  the 
spindle,  are  connected  by  interzonal  filaments  (figs.  29,  30).  "  Zwi- 
schenkorperchen "  form  midway  between  the  ends  of  the  filaments,  as 
described  for  the  first  spindle  (p.  32),  and  later  fuse  into  a  cell  plate. 

The  second  spindles  do  not  differ  from  one  another  much  in  size, 
nor  do  their  dimensions,  on  the  average,  change  appreciably  with  pro- 
longed existence  due  to  the  absence  of  semination.  This  constancy  in 
size  is  shown  by  a  comparison  of  spindles  from  two  groups  of  eggs :  one 
group  composed  of  eggs  which  have  been  but  a  short  time  in  the  oviduct 
(taken  not  later  than  i6j  hours  after  parturition), the  other  of  eggs  taken 
from  the  oviduct  29  or  more  hours  after  parturition.  Because  of  the  unfa- 
vorable position  of  many  spindles,  measurements  of  only  30  young  and  26 
old  ones  could  be  used.  The  average  dimensions  for  the  young  spindles 
are:  length  17.9  micra,  diameter  7.2  micra;  for  the  old  spindles:  length 
17.5,  diameter  7.3  micra.  A  comparison  of  these  averages  with  those  of 
the  mature,  or  nearly  mature,  first  spindles  in  Stages  IVa  and  IVb  (viz., 
19.2  X  10.8  micra,  and  22.4  X  9.9  micra,  respectively)  proves  that  the 
second  maturation  spindle  is  somewhat  smaller  than  the  first. 

4.  CENTROSOMES,  CIRCUMPOLAR  BODIES,  AND  CLEAR  REGION. 

For  the  second  spindle,  as  for  the  first,  the  existence  of  typical 
centrosomes  is  highly  improbable.  However,  there  are  at  certain  times 
structures  which  to  some  extent  resemble  centrosomes. 

The  circumpolar  bodies  (figs.  22,  24)  correspond  exactly  in  posi- 
tion, abundance,  and  general  appearance  to  those  of  the  first  spindle. 
When  the  spindle  is  first  fully  formed  they  are  already  present,  and  per- 
sist for  some  time.  But  with  spindles  which,  in  the  absence  of  semina- 
tion, persist  for  a  long  time  they  have  a  tendency  to  dwindle  away, 
sometimes,  however,  leaving  a  few  granules  at  the  poles  where  centro- 
somes might  be  expected  (figs.  25,  26,  27).  These  statements  are  based 
upon  a  comparison  of  the  eggs  used  in  calculating  the  size  of  the  spin- 
dle. The  eggs  in  the  oviduct  (and  a  few  in  the  periovarial  space  and 
in  the  ovary)  taken  from  mice  killed  16^  hours  or  less  p.p.  show  in  most 
instances  well-developed  circumpolar  bodies,  whereas  most  of  the  eggs 
from  animals  killed  29  or  more  hours  p.p.  show  very  few  or  none  of  them. 
These  bodies  also  disappear  following  normal  metakinesis  induced  by 
semination  after  the  chromosomes  have  migrated  and  become  confluent 
(figs.  29a,  30),  as  in  the  case  of  the  first  spindle. 


40     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

In  some  of  the  eggs  from  a  mouse  killed  about  33  hours  p.p.  and 
not  inseminated,  there  appear  a  few  cytoplasmic  radiations  at  the  spindle 
poles,  from  which  the  circumpolar  bodies  have  vanished.  Also  in  the 
egg  illustrated  in  fig.  2Qa,  the  cytoplasmic  granules  around  the  inner 
end  of  the  spindle  are  oriented  with  their  long  axes  in  a  radial  direction ; 
but  otherwise  the  evidence  of  cytoplasmic  radiations  about  the  poles 
of  the  second  maturation  spindle  has  been  lacking. 

As  already  stated  regarding  the  first  spindle,  the  clear  region  around 
the  spindle  exists  at  the  same  time  with  the  circumpolar  bodies,  except 
that  it  may  appear  a  little  before  them  (fig.  1 9) ,  and  sometimes  persists 
longer  (fig.  30).  It  can  usually  be  found  surrounding  the  spindles  of 
eggs  which  have  been  only  a  short  time  in  the  oviduct  (figs.  20,  21,  22, 
23,  24),  but  in  most  eggs  in  which  the  circumpolar  bodies  have  vanished, 
it  has  likewise  disappeared  (figs.  25,  26,  27).  It  becomes  quite  faint 
(figs.  29,  30),  or  is  altogether  gone,  after  the  chromosomes  have  divided. 
5.  POSITION  AND  ORIENTATION  OF  SECOND  MATURATION  SPINDLE. 

The  second  maturation  spindle  always  lies  near  the  surface  of  the 
egg — in  fact,  sometimes  so  near  that  its  flatness  (fig.  20)  is  apparently  due 
to  pressure.  There  is  no  satisfactory  evidence  that  it  moves  through 
the  cytoplasm,  although  it  is  found  at  different  distances  from  the  first 
polar  cell  when  that  is  present.  This  topic  will  be  taken  up  later  (pp.  44 
and  63). 

There  is  less  variation  in  the  orientation  of  the  second  spindle 
than  in  that  of  the  first.  Very  rarely,  indeed,  can  it  be  found  perpendic- 
ular to  the  surface;  occasionally  it  is  oblique,  but  in  the  majority  of 
cases  it  is  parallel  to  the  surface.  It  is  parallel  in  all  instances  in  which 
the  daughter  chromosomes  have  separated  and  have  reached,  or  nearly 
reached,  the  poles  before  the  abstriction  of  the  second  polar  cell  begins; 
but  in  those  in  which  the  abstriction  has  begun  (figs.  29a,  296)  it  is 

oblique ;  and  in  the  stage  of  the  telophase 
of  the  formation  of  the  second  polar 
cell  (fig.  30)  the  interzonal  filaments  are 
usually  almost ,  if  not  quite ,  perpendicular. 


6.  ABSTRICTION  OF  SECOND  POLAR  CELL. 

The  process  resulting  in  the  forma- 
Fig.  29 a\      'V^^BJBJiil       tion  of  the  second  polar  cell  is  precisely 
5     ^X^V  :'-f^y*C.y       like  that  by  which  the  first  polar  cell  is 
FIG.  J.  produced.    The  beginning  of  the  process 

is  illustrated  by  an  egg  shown  in  part 

in  figs.  2ga  and  296  and  in  fig.  /.  The  last  is  a  diagrammatic,  imaginary 
section  of  the  egg,  in  a  plane  parallel  with  the  axis  of  the  spindle,  but 
perpendicular  to  the  actual  sections  shown  in  figs.  29a  and  296.  The 
daughter  chromosomes  have  virtually  reached  the  poles  of  the  spindle 
and  have  lost  their  identity  by  being  merged  together;  the  "  Zwischenkor- 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  41 

perchen  "  now  occupy  the  middle  of  the  interzonal  filaments.  The  spindle 
is  oblique  to  the  surface  of  the  egg,  and  one  pole  is  so  near  the  surface 
(fig.  2 go)  that  the  peripheral  mass  of  chromatin  lies  close  to  the  edge  of 
the  protrusion  which  is  destined  to  be  cut  off  to  form  the  polar  cell.  The 
constriction  has  begun  on  the  side  nearest  the  "Zwischenkorperchen," 
the  vitelline  membrane  being  already  in  contact  with  the  "  Zwischenkor- 
perchen" nearest  the  surface  of  the  egg  (fig.  296).  The  rest  of  the 
process,  involving  the  final  separation  of  the  polar  cell,  is  as  described 
on  page  34  for  the  first  polar  cell. 

C.  RIPE  EGG. 

STAGE  XI. — THE  PRONUCLEI. 

A  discussion  of  the  further  development  of  the  ripe  egg  does  not 
lie  within  the  scope  of  the  present  work.  It  suffices  to  say  that  the  chro- 
matin mass  resulting  from  the  union  of  the  chromosomes  remaining 
after  the  formation  of  the  second  polar  cell  is  quickly  transformed  into 
the  egg  nucleus.  This  usually  occurs  simultaneously  with  the  develop- 
ment of  the  sperm  nucleus.  But  in  two  cases  the  egg  nucleus  had  reached 
a  diameter  of  6  micra,  while  the  head  of  the  spermatozoon  had  not  been 
appreciably  changed  in  form  or  size.  In  no  case  has  the  sperm  nucleus 
been  observed  before  the  chromatin  mass  has  begun  to  be  transformed 

into  the  egg  nucleus. 

D.  POLAR  CELLS. 

The  observations  on  the  polar  cells  here  recorded  do  not  extend  to 
the  cleavage  stages  of  the  egg.  Therefore,  no  statement  can  be  made 
concerning  the  further  fate  of  the  polar  cells,  or  concerning  the  changes 
which  take  place  in  the  second  polar  cell. 

FIRST  POLAR  CELL. 

The  first  polar  cell,  originating  as  described  on  page  34,  is  usually 
an  ellipsoidal  or  a  flattened  spheroidal  body,  the  three  diameters  of  which 
are  nearly  always  unequal.  The  average  dimensions  of  28  polar  cells — 
each  of  which  had  been  recently  formed  (Stage  VI),  the  first  spindle 
being  still  in  the  telophase  (plate  4,  fig.  18) — were  22.7  X  19.2  X  13.5 
micra.  These  figures  indicate  the  average  size  at  its  largest  stage.  With 
age  some  polar  cells  diminish  very  rapidly  in  size  (figs.  18,  32—37,  plate  6) ; 
others  retain  nearly  their  original  dimensions.  Disregarding  for  the  pres- 
ent the  very  small  forms  (figs.  3 5-3 7),  it  is  found  that  the  first  50  polar 
cells  (which  could  be  measured  most  accurately)  from  100  of  the  young- 
est eggs  which  have  the  complete  second  spindle  give  as  an  average  the 
following  dimensions  in  micra:  20  X  15.6  X  n.8;  and  22  polar  cells 
(all  that  could  be  measured)  from  100  of  the  oldest  eggs  of  the  same 
stage  give  the  following  average  dimensions:  16  X  13  X  10.5  micra. 
These  averages  show  a  considerable  decrease  in  size;  and,  as  a  series  of 
gradually  diminishing  sizes  can  be  found  down  to  that  shown  in  fig.  37, 
and  as  the  smaller  sizes  are  too  numerous  (55  out  of  507  eggs)  to  be  mere 


42     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

chance  occurrences,  it  must  necessarily  follow  that  the  first  polar  cell 
may,  in  many  cases  does,  dwindle  to  almost  nothing.  Indeed,  it  may 
even  disappear  completely;  for  out  of  the  507  eggs  with  complete  second 
spindle,  189  have  no  polar  cell.  This  is  made  clearer  still  when  the  200 
eggs,  mentioned  above,  are  examined  further.  The  results  are  most 
conveniently  presented  in  tabular  form  (table  5).  This  shows  that  of 
the  older  eggs,  as  compared  with  the  younger  ones,  fewer  have  the  large 
polar  cells  and  more  have  no  polar  cell.  The  fewer  cases  with  small 
polar  cell  among  the  older  eggs  show  that  most  of  the  polar  cells  which 
degenerate  do  so  early,  being  completely  wanting  in  the  later  epochs. 
The  same  conclusions  are  borne  out  by  the  162  eggs  of  Stages  IX  to 
XI  (table  2,  p.  14),  which,  as  a  whole,  cover  a  longer  period.  Of  these 
162  eggs,  77  have  no  polar  cell,  22  have  a  small  polar  cell,  and  63  the 
larger  sizes  of  polar  cell. 

TABLE  5. 


Young. 

Old. 

Eggs  with  large  first  polar  cell  ....          .... 

72 

2  C 

Eggs  with  small  polar  cell  (figs.  36,  37)  .... 
Eggs  with  no  polar  cell 

14 

14. 

8 
67 

Totals  

IOO 

IOO 

The  first  polar  cell  contains  the  peripheral  group  of  chromosomes, 
which  have  become  compacted  into  a  single,  usually  flattened  mass 
(plate  4,  fig.  1 8).  During  the  formation  of  the  second  spindle  this  mass 
divides  into  irregular  parts  (fig.  i go),  which  remain  more  or  less  in  conti- 
nuity with  one  another.  It  is  only  rarely  that  these  parts  separate  from 
one  another  completely  and  assume  the  aspect  of  dumb-bell  shaped  bodies. 
Their  number,  however,  has  no  significance,  owing  to  their  imperfect 
form  and  individuality.  The  chromatin  may  remain  for  a  considerable 
period  in  one,  or  more  than  one,  loosely  formed  mass.  If  it  is  more  finely 
divided,  the  fragments  may  be  distributed  with  tolerable  uniformity 
throughout  the  cytoplasm  (plate  5,  figs.  30,  31  a),  or  roughly  aggregated 
into  two  groups,  one  at  each  end  of  the  cell.  Not  infrequently  the  chro- 
matin bodies  exhibit  thread-like  forms, especially  in  connection  with  what 
appears  otherwise  to  be  a  non-mitotic  division  of  the  polar  cell  (figs.  3  2, 3  3). 
In  no  case,  however,  has  it  been  observed  that  the  chromatin  is  drawn 
to  the  equator  of  a  well-formed  spindle  and  divided.  Often  the  chromatin 
fragments,  especially  the  enlarged  ends  of  the  thread-like  forms,  show 
vacuolation  (figs.  30,  32,  33).  Besides  the  deeply  staining  chromosomal 
bodies,  there  are  other  less  deeply  staining  bodies  (figs.  296,  32,  33,34,35), 
which  apparently  are  modified  chromatin;  these  occur  either  alone — 
especially  is  this  the  case  in  small  polar  cells  (figs.  34,  35) — or  associated 
with  vacuolating  parts  (figs.  32,  33).  These  conditions  all  seem  to  point 
to  a  degeneration  of  the  chromatin.  A  nucleus  is  never  formed,  unless 


OBSERVATIONS    ON    THE    MATURATION    PROCESSES.  43 

perhaps  it  arises  in  divided  first  polar  cells  during  the  cleavage  stages  of 
the  egg. 

Although  the  cytoplasm  of  the  polar  cell  has  not  been  studied  care- 
fully by  us,  its  general  features  are  as  follows.  In  the  newly  formed 
polar  cell  the  more  distal  part  of  the  cytoplasm  appears  very  clear  (figs. 
1 8,  19).  Later,  it  is  of  uniform  appearance  throughout  the  cell,  and  in 
some  cases  is  apparently  like  that  of  the  egg ;  but  more  often  it  is  either 
more  granular  or  more  homogeneous  and  clear  than  the  egg  cytoplasm. 
In  the  smaller  polar  cells  it  has  the  latter  structure  and  it  sometimes 
shows  what  appear  to  be  ill-defined  vacuoles  (fig.  36).  The  interzonal 
filaments  within  the  polar  cell  are,  at  first,  very  evident  (figs.  18,  19).  In 
time  they  lose  their  connection  with  the  cell  plate  (figs.  1 9  and  3  ia) ,  which 
then  quickly  disappears.  Occasionally  there  can  be  observed  in  the  polar 
cell  fibers  which  are  parallel  with  one  another;  but  it  is  uncertain  whether 
they  are  the  remains  of  interzonal  filaments  or  fibers  of  an  abortive 
spindle. 

It  may  be  inferred  from  the  amitotic  (or  imperfect  mitotic)  division 
of  the  chromatin  that  the  whole  polar  cell  is  capable  of  division.  Such, 
indeed,  is  the  case,  for,  previous  to  the  formation  of  the  second  polar 
cell,  the  first  polar  cell  may  be  observed  in  many  instances  to  be  divid- 
ing into  two  or  more  parts,  as  shown  in  figs.  32  and  33,  or  to  be  simply 
constricted  (fig.  31  a).  Less  frequently  the  small  polar  cell  is  seen  to  be 
already  divided  into  two  parts.  This  dividing  of  the  polar  cell  doubt- 
less aids  in  its  rapid  degeneration  by  increasing  the  external  surface 
exposed  to  the  action  of  absorption. 

The  polar  cell  quickly  loses  its  connection  with  the  egg,  because  the 
interzonal  filaments  become  severed  from  the  cell  plate.  This  separa- 
tion is  evident  as  early  as  the  time  of  ovulation  and  may  be  aided  by 
that  process,  as  described  on  page  22  and  shown  in  figs.  3ia  and  316,  38, 
39,  and  40  (figs.  3ia  and  316  being  enlarged  views  of  sections  of  the  egg 
and  polar  cell  of  which  fig.  40  shows  another  section).  In  the  egg  illus- 
trated in  figs.  3ia  and  316  the  polar  cell  is  separated  from  the  egg  and 
probably  from  the  cell  plate,  which  is  seen  in  fig.  316.  (In  this  case, 
however,  the  existence  of  the  cell  plate  is  a  little  doubtful.)  The  evi- 
dence leads  to  the  belief  that  the  first  polar  cell  need  not  remain  at  the 
place  where  it  was  formed,  but  may,  according  to  circumstances,  change 
its  position  under  the  zona,  even  to  such  an  extent  as  to  come  to  lie 
diametrically  opposite  the  point  of  its  origin.  The  bearing  of  these 
observations  on  the  question  of  the  relative  positions  of  the  first  polar 
cell  and  the  second  spindle  will  be  considered  later  (p.  63).  The  first 
polar  cell  usually  lies  in  a  depression  in  the  surface  of  the  egg. 


44  THE    MATURATION    OF    THE    EGG    OF    THE    MOUSE. 

SECOND  POLAR  CELL. 

The  shape  of  the  second  polar  cell  is  similar  to  that  of  the  first, 
though  it  is  perhaps  more  often  uniformly  regular  in  shape.  In  order 
to  compare  the  size  of  the  second  polar  cell  with  that  of  the  first,  meas- 
urements were  made  of  as  many  newly  formed  polar  cells  as  possible 
(Stage  X,  fig.  30).  Since  the  condition  of  the  polar  cells  during  cleavage 
stages  of  the  egg  has  not  been  studied,  changes  in  size  are  not  here  con- 
sidered. For  convenience  the  sizes  of  the  first  polar  cells  (exclusive  of 
the  small  degenerate  forms)  are  repeated  in  this  connection  (table  6). 

TABLE  6. — Size  of  polar  cells. 
Average  dimensions  of  first  polar  cell.  Micra. 

Newly  formed  polar  cell  (first  spindle  in 

telophase) 22  .7X19.2  X  13  .5 

From  eggs  but  a  short  time  in  the  oviduct 

(complete  second  spindle) 20  Xi5.6Xn.8 

From  eggs  after  29  hours  in  the  oviduct.  ..    16      Xi3      Xio.5 

Average  dimensions  of  second  polar  cell. 
Newly  formed 19 . 3X16. 7  X   9.6 

When  first  produced  the  second  polar  cell,  then,  is  smaller  than  the 
first  polar  cell  of  corresponding  age,  but  is  larger  than  the  first  polar  cell 
which  has  been  in  existence  for  29  hours  or  more. 

At  the  beginning,  the  chromatin  of  the  second  polar  cell  is  in  a 
single  mass,  as  in  the  case  of  the  first  polar  cell,  but  it  does  not  long 
remain  so,  for  it  is  quickly  transformed  into  a  nucleus. 

The  cytoplasm  in  the  recently  cut  off  cell  (fig.  30)  has  the  clear 
appearance  noted  in  the  case  of  the  first  polar  cell,  but  later  it  generally 
has  the  aspect  of  the  protoplasm  of  the  egg.  The  interzonal  filaments 
persist  for  a  time  and  can  be  observed  joining  the  nucleus  of  the  polar 
cell  with  that  of  the  egg,  the  cell  plate  remaining  as  a  conspicuous, 
deeply  stained  body  outside  both  egg  and  polar  cell. 

The  position  of  the  second  polar  cell  with  regard  to  the  first  (when 
the  latter  is  present)  is  variable,  for  the  two  polar  cells  may  lie  side 
by  side  or  be  far  apart.  The  reason  is  probably  to  be  found  in  the  migra- 
tion of  the  first  polar  cell,  as  discussed  on  page  63.  The  second  polar 
cell,  like  the  first,  occupies  a  slight  depression  in  the  surface  of  the  egg. 


CRITICISMS    AND    CONCLUSIONS.  45 

VIII.  CRITICISMS  AND  CONCLUSIONS. 
A.  MATERIAL. 

This  work  differs  from  that  of  previous  investigators  in  that  it  has 
been  done  on  mice  of  very  mixed  ancestry.  It  is  therefore  open  to  the 
possible  criticism  that  the  material  is  unlike  that  on  which  other  papers 
have  been  based.  It  may  be  maintained,  however,  that  there  is  no 
essential  difference  in  material  for  the  following  reasons:  first,  the  fact 
that  the  white  mice  of  our  stock,  whether  of  colored  ancestry  or  not, 
breed  true,  leads  one  to  believe  that,  in  the  light  of  recent  work  on  hered- 
ity of  coat  color,  they  are  as  pure  as  other  white  mice;  secondly,  there 
is  no  dissimilarity  in  the  maturation  processes  of  eggs  from  mice  of  dif- 
ferent coat  character;  thirdly,  there  is  no  real  difference  in  important 
points  between  Mr.  Kirkham's  preparations  and  our  own. 

Sobotta  suggests  in  his  paper  published  in  1907  that  some  of  the 
differences  between  his  results  and  those  of  Gerlach  (1906)  may  be  due, 
in  part  at  least,  to  the  fact  that  he  used  eggs  set  free  at  an  ovulation  3 
weeks  after  parturition,  whereas  Gerlach  employed  ova  obtained  during 
the  first  3  days  after  parturition.  Since  Sobotta  is  the  only  one  who  has 
made  use  of  eggs  derived  from  an  ovulation  later  than  the  first  one  after 
the  birth  of  young,  his  explanation  must  apply  to  all  other  investigations, 
including  the  present  one.  There  seems,  however,  to  be  no  a  priori 
reason  for  supposing  a  difference  between  the  maturation  processes  of 
eggs  maturing  and  ready  for  fertilization  at  different  periods  after  par- 
turition; moreover,  the  dissimilarities  in  the  results  of  the  several  in- 
vestigators can  be  accounted  for  to  a  large  extent  on  other  grounds,  as 
will  appear  in  the  course  of  the  remaining  pages. 

Considerable  significance  attaches  to  the  amount  of  material  studied 
by  other  investigators.  Tafani,  Gerlach,  and  Kirkham  do  not  state  the 
number  of  eggs  which  they  observed,  but  the  number  was  probably 
small.  Lams  et  Doorme  based  their  paper  on  only  90  ova.  Sobotta  in 
his  large  work  (1895)  used  1402  sound  eggs;  but  of  this  number  only  298 
(compared  with  our  877,  table  2,  Stages  I-X),  at  the  most,  were  of 
such  age  as  to  show  stages  in  the  formation  or  division  of  spindles,  or 
the  number  of  chromosomes,  or  the  abstriction  of  polar  cells.  All  the 
rest  (1104)  were  either  in  stages  showing  the  pronuclei  or  still  older. 

B.  METHODS. 

It  is  probable  that  the  value  of  our  results  would  have  been  en- 
hanced had  another  set  of  eggs  preserved  by  other  methods  been  com- 
pared at  each  step  with  those  which  have  served  as  the  basis  for  the 
present  paper.  These  have  all  been  carefully  studied  and  in  part  are 
described  and  figured  here.  However,  it  should  be  said  that  other  fix- 
ing fluids  were  tried,  and  that,  in  cases  where  the  preservation  was  good 
enough  to  give  reliable  pictures,  the  eggs  showed  conditions  similar  to 
those  obtained  with  the  special  preserving  fluid  described  at  page  12. 
4 


46     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

This  modification  of  Zenker's  fluid,  however,  is  the  only  one  tried  which 
shows  the  finer  structure  of  the  chromosomes  and  does  not  shrink  the 
nuclei.  Most  of  the  figures  by  other  investigators  of  the  mouse  egg 
show  imperfect  preservation,  and  this  has  been,  in  our  opinion,  a  potent 
factor  in  causing  the  differences  in  their  results. 

C.  TIME  RELATIONS. 

The  possibility  of  obtaining  a  complete  series  of  stages  of  the  proc- 
esses of  maturation  depends  on  accuracy  in  determining  the  epochs 
of  parturition  and  insemination.  As  we  have  seen  (p.  22),  there  is 
probably  some  individual  variation  in  the  length  of  the  periods  between 
successive  ovulations.  If  eggs  from  ovulations  other  than  the  one  which 
immediately  follows  parturition  had  been  used  by  us,  it  would  have  been 
extremely  difficult,  if  not  impossible,  to  secure  a  complete  series,  both 
because  of  this  variability  in  ovulation,  and  also  because  (see  p.  17) 
the  stage  of  the  second  spindle  may  last  for  many  more  hours  than  the 
stages  which  reach  from  the  transformation  of  the  germinative  vesicle 
to  the  formation  of  the  first  polar  cell.  It  is  probably  a  lack  of  precision 
in  this  matter  which  accounts  for  the  failure  of  others  to  get  those  stages 
which  pass  quickly,  such,  for  example,  as  the  origin  and  metaphase  of 
the  first  spindle. 

As  we  have  seen  (pp.  16  and  19),  the  first  maturation  after  parturi- 
tion may  occur  during  a  period  extending  from  about  13  hours  to  29 
hours  p.p.  Tafani  (1889,  p.  20)  makes  the  period  extend  from  24  hours 
to  48  hours,  or  even  (18896,  p.  113)  2  or  3  days  p.p.,  a  time  somewhat 
later  than  that  indicated  by  our  observations. 

Sobotta  (1907,  p.  504)  says  that  the  prophases  of  the  first  spindle 
begin  at  least  24  hours  before  ovulation;  but  as  he  does  not  say  when 
ovulation  occurs  with  respect  to  parturition  (which  is  the  only  event 
that  can  be  determined  directly),  it  is  impossible  to  perceive  how  he 
arrives  at  this  particular  number  of  hours  as  the  minimum  time.  Appar- 
ently Sobotta  (p.  507)  bases  this  conclusion  on  the  parallelism  which, 
he  maintains,  exists  between  the  histological  changes  in  the  wall  of  the 
follicle  (its  ripening)  and  the  ripening  of  the  egg ;  but  admitting  the  paral- 
lelism, and  granting  that  the  prophase  begins  when  the  follicle  is  far 
from  ripe,  we  are  unable  to  see  any  very  precise  ground  for  the  estimated 
time  required  for  the  ripening  of  the  follicle. 

Kirkham  (1907(3,  p.  259)  states  that  he  killed  mice  at  various  times 
during  pregnancy  and  at  intervals  from  a  few  minutes  after  parturition 
up  to  30  hours  after  that  event.  In  his  later  paper  (19076,  pp.  70,  71) 
he  adds: 

The  ovaries  of  every  mouse  examined  during  the  height  of  the  breeding  season 
contained  some  eggs  in  which  the  first  polar  body  had  been  already  extruded  and 
in  which  the  spindle  for  the  second  polar  mitosis  was  fully  formed.  A  majority  of 
the  same  ovaries  revealed  ovarian  eggs  at  the  end  of  the  spireme  or  with  the  first 
polar  spindle. 


CRITICISMS    AND    CONCLUSIONS.  47 

Also  (p.  75): 

A  large  number  of  eggs  in  different  ovaries  have  been  examined,  and  in  every 
instance  where  the  size  of  the  egg,  its  slightly  denser  protoplasm,  and  the  large  folli- 
cle gave  evidence  of  ripeness,  the  egg  was  found  to  be  accompanied  by  the  first  polar 
body.  This  agrees  with  the  observations  of  Bellonci  (1885),  and  with  Sobotta's 
idea  regarding  10  per  cent  of  the  eggs,  which  he  believed  formed  two  polar  bodies. 

These  two  statements  appear  at  first  sight  either  to  relate  to  differ- 
ent stages  of  maturation  or  else  to  be  difficult  to  reconcile  with  each 
other;  but  further  consideration  leads  us  to  think  that  the  same  condi- 
tions are  intended  in  both.  According  to  the  first  quotation,  a  part  of 
the  more  advanced  eggs  are  only  just  beginning  maturation  (spireme 
or  first  spindle),  while  others  are  further  along,  showing  the  first  polar 
cell  and  second  spindle.  In  the  second  quotation  only  the  older  eggs, 
those  with  the  first  polar  body,  are  mentioned;  but  it  is  perhaps  fair  to 
infer  that  here,  too  (as  announced  in  the  first  statement  quoted),  others 
were  just  beginning  the  process  of  maturation,  though  it  is  explicitly 
stated  that  "in  every  instance"  the  first  polar  body  was  present.  How- 
ever that  may  be,  it  is  clearly  stated  that  in  every  mouse  examined 
during  the  height  of  the  breeding  season  the  ovary  contained  some  eggs 
which  showed  the  first  polar  cell  and  the  second  spindle.  Since  the  author 
certainly  studied  and  figured  (his  figs.  12—17)  eggs  from  the  Fallopian 
tube,  it  is  impossible  to  avoid  the  inference  that  in  all  females,  even  in 
those  in  which  one  set  of  eggs  is  in  the  oviduct,  the  ovaries  contain  eggs 
with  the  first  polar  cell  and  the  second  spindle  already  formed;  that  is 
to  say,  maturation  may  begin  several  weeks  before  parturition  or  ovu- 
lation.  But  such  a  state  of  affairs  is  incomprehensible  to  us,  because, 
according  to  our  studies,  mice  killed  during  pregnancy  and  at  intervals 
of  7  and  14  days  after  parturition  furnished  ovarian  eggs  (these  have 
not  been  included  in  the  1,000  eggs  recorded  in  table  2)  some  of  which 
were  in  fairly  large  follicles.  Those  in  the  largest  follicles  (eggs  which 
presumably  were  destined  to  leave  the  ovary  at  the  next  ovulation) 
possessed  in  all  cases  the  germinative  vesicle.  Such  was  also  the  case  in 
mice  killed  during  a  period  extending  from  i  to  13  hours  after  partu- 
rition. Eggs  with  the  germinative  vesicle,  which,  as  has  already  been 
explained  (p.  16),  do  not  acquire  the  first  spindle  before  about  13  hours 
post  partum,  manifestly  could  not  originate  by  the  transformation  of 
eggs  already  possessing  a  polar  cell  and  second  spindle.  Moreover, 
mice  which  showed  a  group  of  eggs  in  each  oviduct  never  exhibited  any 
of  the  large  follicles  in  the  ovary.  Lastly,  as  has  already  been  demon- 
strated (p.  15),  only  two  mice  furnished  eggs  in  stages  as  widely  separated 
as  those  of  the  germinative  vesicle  and  of  the  first  polar  cell  and  second 
spindle ;  and  in  these  two  cases  the  eggs  exhibiting  the  early  stage  were 
in  one  ovary,  while  the  eggs  showing  the  later  stage  were  in  the  oviduct 
of  the  other  side  of  the  body.  At  first  the  only  explanation  of  the  differ- 
ences between  Dr.  Kirkham's  results  and  our  own  which  seemed  to  us 
possible  was  that  his  mice  were  of  a  different  breed  from  ours. 


48     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

Through  the  kindness  of  Professor  Coe,  of  Yale  University  (Dr. 
Kirkham  being  abroad) ,  we  had  the  privilege  of  examining  a  portion  of 
Dr.  Kirkham's  preparations,  some  25  slides,  on  which  the  position  of 
eggs  with  first  polar  cell  and  second  spindle  and  that  of  eggs  with  a 
single  spindle  had  been  marked  by  the  author.  An  examination  of  these 
preparations  revealed  the  fact  that -nearly  all  of  the  ovarian  eggs  so 
marked  were  in  process  of  degeneration.  They  were  of  about  normal 
size,  but  occurred  in  rather  small  follicles,  approximately  like  the  one 
shown  in  Kirkham's  (19076)  plate  V,  fig.  n.  The  zona  pellucida  was 
gone,  and  the  granulosa  cells  were  only  rarely  in  contact  with  the  egg — 
sure  signs,  in  our  opinion,  of  degeneration.  Such  eggs  can  be  found  in 
nearly  all  ovaries;  but  we  have  always  rigidly  excluded  them,  because 
they  are  so  obviously  different  from  the  normal  eggs  contained  in  the 
large  follicles.  Sometimes  in  these  small  follicles  there  can  be  found 
clusters  of  cells  resulting  apparently  from  the  abnormal  cleavage  of  de- 
generating egg  cells.  These  facts  explain,  we  think,  fig.  7  of  Kirkham's 
second  paper  (19076),  a  figure  which  Sobotta  (1908,  p.  260)  could  not 
understand,  and  also  fig.  n  of  the  same  paper,  which  is  clearly  that 
of  a  degenerating  egg.  Kirkham  (19076,  p.  77)  says,  in  explanation  of 
the  absence  of  the  zona  from  this  and  all  other  eggs  of  the  same  series 
(presumably  the  same  animal),  that  it  is  "probably  due  to  the  solvent 
action  of  the  killing  fluid."  But  it  certainly  would  be  remarkable  if 
the  same  killing  fluid  operated  so  differently  on  different  ovaries.  The 
explanation  which  we  have  suggested — a  degenerating  condition  of  the 
ova — is  rendered  still  more  probable  by  the  fact  that  "all  the  ovarian 
eggs  in  this  series  are  likewise  naked."  Tafani  (1889,  p.  24)  in  his  criti- 
cism of  Bellonci  expresses  the  opinion  that  the  latter  saw  in  degenerating 
follicles  eggs  which  never  would  have  been  set  free,  but  which  formed 
polar  cells.  Such  eggs  are  just  what  Bellonci,  having  little  material, 
would  probably  have  seen  and  misinterpreted,  for  the  reason  that  they 
occur  in  all  ovaries  of  mature  mice  at  all  times,  whereas  normal  eggs  con- 
taining the  first  spindle  or  the  first  polar  cell  and  second  spindle  can  be 
found  only  during  a  very  limited  period.  However,  it  must  be  borne 
in  mind  that,  while  Tafani  did  not  misinterpret  degenerating  eggs,  he 
did  confuse  the  first  and  second  spindles.  He  saw  the  first  spindle  in 
the  ovarian  egg,  but  apparently  not  the  formation  of  the  first  polar  cell, 
and  seeing  a  spindle  (the  second)  in  eggs  in  the  oviduct  without  the  first 
polar  cell,  he  mistook  it  for  the  first  spindle.  That  he  missed  the  stage 
of  the  abstriction  of  the  first  polar  cell  is  rendered  the  more  probable 
by  the  fact  that  he  placed  the  period  of  maturation  rather  late  and  studied 
so  many  eggs  from  the  oviduct.  Nevertheless,  Tafani's  criticism  of 
Bellonci  was  probably  sound. 

There  are  apparently  no  statements  in  any  of  the  works  on  the  em- 
bryology of  mammals  which  show  precisely  how  much  time  is  required 
for  any  part,  or  the  whole,  of  the  maturation  process.  Indeed,  the 


CRITICISMS    AND    CONCLUSIONS.  49 

length  of  time  required  in  the  mouse  according  to  our  observations, 
namely,  from  4  to  15  hours,  needs  confirmation. 

According  to  the  calculations  of  Tafani  (18896,  p.  114)  the  interval 
between  coitus  and  the  penetration  of  the  spermatozoon  is  7  or  8  hours, 
of  Sobotta  (1895,  p.  63)  and  Gerlach  (1906,  p.  8)  6  to  10  hours.  Tafani 
and  Sobotta  think  the  formation  of  the  pronucleus  requires  only  about 
an  hour  from  the  time  the  spermatozoon  penetrates  the  egg;  whereas 
Gerlach  does  not  believe  the  pronucleus  is  formed  so  quickly.  We  have 
already  (p.  21)  shown  that  the  interval  between  coitus  and  penetration 
may  be  much  less,  viz,  4  to  7  hours,  and  that  the  pronuclei  probably 
require  only  a  few  minutes  for  their  development. 

D.  OVULATION. 

It  is  desirable  to  know  whether  the  time  of  ovulation  has  any  fixed 
relation  to  that  of  either  coitus  or  parturition. 

All  investigators  except  Gerlach  (1906,  p.  22)  agree  that  in  the 
mouse  ovulation  is  independent  of  coitus,  although  such  is  not  the  case 
in  some  other  mammals,  e.g.,  the  rabbit  and  the  guinea-pig.1  Regarding 
the  relation  of  ovulation  to  parturition,  Kirkham  (19076,  p.  79)  is  the 
only  one,  so  far  as  we  know,  who  makes  any  statement.  He  says  that 
ovulation  takes  place  in  from  i  to  2  hours  after  parturition;  but  as  he 
cites  no  authority  for  the  statement  and  furnishes  no  evidence  of  his 
own,  one  can  not  give  his  conclusion  much  weight.  We  have  already 
given  evidence  that  it  occurs  at  some  time  during  a  period  extending 
from  14^  to  28^  hours  after  parturition. 

There  is  some  difference  of  opinion  concerning  the  relation  of  the 
time  of  ovulation  to  that  of  maturation,  the  chief  cause  of  which  seems 
to  us  to  be  the  failure  to  find  any  critical  basis  for  distinguishing  between 
the  first  and  the  second  maturation  spindles.  Tafani  (1889,  p.  22)  says 
ovulation  occurs  during  the  stage  of  the  first  spindle.  While  this,  in 
our  opinion,  is  not  true,  the  statement  can  be  explained  on  the  highly 
probable  assumption  that  he  confused  the  first  and  second  spindles. 
Sobotta  has  changed  his  opinion  since  writing  in  1895,  and  now  (1907, 
pp.  515,  519,  546;  1908,  pp.  247,  250)  believes  that  ovulation  occurs  only 
during  the  monaster  stage  of  the  second  spindle.  He  never  finds  the  first 
spindle  in  eggs  encountered  in  the  oviduct,  but  describes,  as  being  found 
in  the  oviduct  (1907,  p.  524,  fig.  8),  what  he  thinks  may  be  a  transition 
stage  between  the  first  and  the  second  spindles.  Gerlach  (1906,  p.  14) 
believes  that  the  changes  in  the  wall  of  the  follicle  that  make  ovulation 
possible  are  not  directly  connected  with  the  maturation  changes  within 
the  egg  itself,  and  therefore  that  the  rupture  of  the  follicle  may  take 
place  at  various  phases  of  maturation ;  but  he  says  that  at  the  earliest  the 
egg  leaves  the  ovary  in  the  stage  corresponding  with  the  beginning  of 
the  first  spindle,  and  at  the  latest  in  that  of  the  second  spindle;  but  this 

1  Cf.  Kirkham,  19076,  p.  79. 


50     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

statement  is  based  on  his  assumption  that  oviducal  eggs  without  polar 
cells  contain  the  first  spindle,  a  view  which  arises  from  his  being  unable 
to  distinguish  between  the  two  spindles  in  the  monaster  stage.  This 
statement  of  Gerlach's  has  been  disproved  by  Sobotta. 

Lams  et  Doorme  (1907,  p.  284)  maintain  that  ovulation  takes  place 
only  during  the  stage  of  the  second  spindle;  but,  as  Sobotta  (1908, 
p.  259)  points  out,  they  contradict  themselves  by  describing  as  a  first 
maturation  spindle  one  found  in  an  ovum  occupying  the  oviduct.  Ac- 
cording to  Kirkham,  the  first  polar  cell  is  always  formed  in  the  ovary; 
but,  as  we  have  seen,  this  statement  is  supported,  in  part  at  least,  by 
false  evidence.  In  spite  of  some  diversity  of  opinion  regarding  the  pre- 
cise state  of  the  egg  at  ovulation,  all  agree  that  ovulation  occurs  during 
the  stage  of  the  second  spindle.  We,  too,  find  this  to  be  generally  but 
not  invariably  true.  It  is  probably  owing  to  the  unusually  large  number 
of  eggs  in  the  earlier  stages  of  maturation  studied  by  us  that  we  have 
found  in  the  periovarial  space  eggs  in  the  stage  of  the  first  spindle,  and 
also  in  the  oviduct  others  that  have  already  formed  the  first  polar  cell 
but  have  not  yet  developed  the  second  spindle.  It  might  be  maintained 
that  these  eggs  had  been  abnormally  retarded  in  their  development, 
and  it  must  be  admitted  that  such  cases  are  not  numerous  enough  to 
allow  one  to  say  that  it  is  a  common  condition.  On  the  other  hand, 
nothing  else  about  these  eggs  pointed  to  their  being  in  any  way  abnormal, 
and  no  signs  of  degeneration  were  discoverable.  These  cases  seem, 
therefore,  simply  to  prove  that  the  general  rule  regarding  the  time  of 
ovulation  in  relation  to  maturation  is  not  so  inflexible  as  one  would 
infer  from  the  observations  hitherto  published. 

E.  SIZE  OF  EGG. 

Sobotta  and  Kirkham  alone  have  published  measurements  of  the 
egg,  Sobotta  on  fixed  material  and  Kirkham  on  living  material.  Sobotta 
(1908)  states  that  ovarian  eggs  before  the  formation  of  the  first  polar 
cell  measure  from  65  to  70  micra  in  diameter,  and  oviducal  eggs  60  micra; 
but  he  does  not  say  what  is  the  average  in  the  former  case,  nor  that  the 
latter  measurement  is  an  average,  though  such  is  presumably  the  case. 
Gerlach  thinks  there  is  considerable  individual  variation,  and  Lams  et 
Doorme  hold  that  oviducal  eggs  are  smaller  than  ovarian  ones.  Our 
conclusions  (see  table  2,  p.  14,  also  p.  24)  substantially  confirm  the  above, 
except  that  the  averages  we  give  are  a  little  less  than  the  dimensions 
published  by  Sobotta.  Kirkham  (19076,  p.  72)  arrives  at  a  different  con- 
clusion, namely,  80  micra  as  the  diameter  of  ovarian  eggs  and  73  to  78 
micra  of  oviducal  eggs;  but  there  may  be  some  doubt  concerning  the 
reliability  of  his  measurements  because  his  methods  may  have  been 
somewhat  faulty,  as  we  shall  explain  directly.  Tafani,  who  was  the  first 
to  study  living  eggs,  carefully  states  (1889,  p.  6)  that  he  collected  them 
from  the  oviduct  and  kept  them  at  the  proper  temperature  in  the  fluid 


CRITICISMS    AND    CONCLUSIONS.  51 

from  the  ovarian  capsule  or  oviduct;  but,  unfortunately,  he  does  not 
give  the  dimensions,  and  his  figures  are  too  diagrammatic  to  serve  as  a 
means  of  determining  size.  Kirkham  has  apparently  overlooked  the 
above  statement,  for  he  says  that  Tafani  makes  no  mention  of  the  method 
used  to  obtain  living  eggs.  Kirkham  (19076,  p.  70)  procures  them  by 
killing  a  female  soon  after  ovulation  is  supposed  to  have  occurred,  re- 
moving the  ovaries  and  Fallopian  tubes  to  a  slide,  and  gently  teasing 
them  with  fine  needles  until  the  eggs  are  seen  to  drop  out ;  he  then  trans- 
fers them  to  the  stage  of  the  microscope  for  study.  Kirkham  does  not 
state  in  what  fluid  he  studied  the  eggs.  The  medium,  however,  is  impor- 
tant, since  it  might,  if  not  like  the  natural  fluid  in  osmotic  action,  either 
swell  or  shrink  the  egg.  We  have  already  shown  that  a  prolonged  stay 
of  eggs  in  the  oviduct  in  the  several  cases  results  in  an  increase  in  their 
size,  the  eggs  used  for  comparison  being  also  subjected  to  precisely  the 
same  treatment  as  those  from  the  oviduct.  Since  Kirkham's  determina- 
tion of  the  time  of  ovulation  is  in  error  by  10  hours  or  more,  it  is  a  little 
doubtful  whether  all  Ms  eggs  were  in  a  normal  condition. 

F.  MATURATION  PROCESSES. 
1.  GERMINATIVE  VESICLE. 

It  is  agreed  by  all  investigators  that  the  germinative  vesicle  is  at 
first  very  near  the  center  of  the  egg,  and  that  it  becomes  more  eccentric 
as  the  time  of  its  transformation  into  the  first  spindle  approaches. 
Tafani  and  Gerlach  both  state  that  its  membrane  becomes  irregular 
and  disappears  soon  after  the  chromosomes  have  begun  to  form. 

2.  FIRST  SPINDLE. 

CHROMATIN. 

Tafani  (1889,  p.  21)  believed  that  by  the  rupture  of  the  germina- 
tive vesicle  the  nucleolus  escaped  as  an  angular  chromatophilous  mass 
and  moved  toward  the  surface  of  the  egg,  where  it  gave  rise  to  the  chro- 
mosomes, while  the  remnants  of  the  vesicle  degenerated  in  the  cyto- 
plasm. We  have  observed  that  the  cluster  of  chromosome  fundaments 
sometimes  has  the  appearance  of  such  an  angular  mass,  and  it  is  possible 
that  Tafani  mistook  this  for  the  nucleolus.  He  figures  it  as  in  the  act 
of  slipping  out  of  the  germinative  vesicle.  In  Sobotta's  opinion  (1895, 
p.  44)  the  chromosomes  in  eggs  which  produce  but  one  polar  cell  are 
formed  from  the  chromatin  of  the  whole  nucleus,  not  merely  from  that 
of  the  nucleolus  as  was  claimed  by  Holl  (1893),  whose  conclusions  are, 
in  Sobotta's  opinion,  unreliable  because  of  the  poor  preservation  of  his 
material.  Sobotta's  statement  (1895,  p.  44)  that  the  chromosomes  are 
very  irregular  in  form  before  they  become  arranged  in  the  equator  of 
the  spindle  and  his  illustration  of  the  condition  (Taf.  4,  fig.  9,  go)  must 
really  relate  to  the  second  spindle,  for  they  are  both  based  on  eggs  from 
either  the  periovarial  chamber  or  the  beginning  of  the  oviduct ;  but  such 
eggs  must  have  already  passed  beyond  the  stage  of  the  first  spindle,  as 


52     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

Sobotta  himself  admits  in  a  more  recent  paper  (1907).  Although  he 
makes  no  mention  of  having  seen  the  beginning  of  the  (large)  first  spindle, 
he  states  (1895,  p.  52;  1907,  p.  507),  without  qualification  or  conclusive 
evidence,  that  it  originates  about  24  hours  before  ovulation.  According 
to  Gerlach  (1906,  p.  9)  the  nucleolus  disappears  completely,  and  from 
the  chromatin  spherules  (which  he  believes  owe  their  origin  to  the  nucleo- 
lus) the  chromosomes  are  differentiated  before  the  disappearance  of  the 
nuclear  membrane.  Kirkham  (19076,  p.  73),  describing  the  prophase 
of  the  first  maturation,  says  that  in  a  few  cases  there  were  traces  of  the 
nuclear  membrane,  though  more  often  it  had  entirely  disappeared.  His 
fig.  i  (plate  I),  though  described  as  that  of  an  ovarian  egg  before  the 
formation  of  the  first  maturation  spindle,  looks  more  like  the  cross- 
section  of  a  spindle  in  the  monaster  stage  than  an  early  stage  in  the  meta- 
morphosis of  the  germinative  vesicle,  and  the  two  detached  chromosomes 
may  possibly  owe  their  peculiar  position  to  the  displacement  which 
sometimes  is  caused  by  the  knife  in  sectioning. 

It  will  be  remembered  (p.  25)  that  the  wall  of  the  nucleolus  is  thick 
and  deeply  stained,  and  that  the  chromatin  bodies  of  the  germinative 
vesicle  are  especially  numerous  around  the  nucleolus,  which  lies  at  one 
side  of  the  vesicle.  Since,  in  the  next  stage,  the  chromosome  fundaments 
(see  p.  26)  are  also  at  one  side  of  the  nucleus,  it  is  probable  that  they 
replace  both  the  vesicular  nucleolus  and  the  chromatin  bodies.  This  is 
rendered  the  more  probable  by  the  fact  that  these  fundaments  are  ar- 
ranged at  one  side  of  a  slightly  denser  part  of  the  nucleoplasm.  Such 
conditions  lead  one  to  think  it  possible  that  the  fundaments  arise  from 
both  the  wall  of  the  nucleolus  and  the  chromatin  bodies,  while  the  achro- 
matic spindle  comes  from  other  parts  of  the  nucleus,  or  possibly  originates 
in  the  inner  part  of  the  nucleolus. 

Precisely  how  the  chromatin  of  the  germinative  vesicle  is  metamor- 
phosed or  differentiated  into  the  fundaments  of  the  chromosomes  is 
unknown ;  but  in  three  cases  the  arrangement  of  the  curved  fundaments 
(as  in  fig.  36)  suggests  the  possibility  that  they  lie  end  to  end  and  may 
therefore  be  regarded  as  parts  of  a  potential  thread  or  spireme.  This 
possibility  is  perhaps  strengthened  by  the  fact  that  these  fundaments 
usually  show  a  longitudinal  division  first  and  the  transverse  division  later. 
These  observations  suggest  that  the  longitudinal  division  may  corre- 
spond to  the  longitudinal  split  in  the  spireme  of  the  synapsis  stage  ob- 
served in  many  invertebrates,  and  that  each  fundament  consists  of  two 
univalent  chromosomes  united  end  to  end.  The  univalent  chromosomes 
would  then  be  sometimes  indicated  by  the  cross- division,  and  would  be 
separated  at  the  first  mitosis,  as  described  on  page  30. 

An  inspection  of  the  figures  of  the  chromosomes  of  the  first  spindle 
in  the  papers  of  Sobotta  (1895,  l899>  I9°7)>  Gerlach  (1906),  Lams  et 
Doorme  (1907),  and  Kirkham  (19076)  reveals  the  fact  that  there  is  no 
essential  disagreement  in  regard  to  the  general  forms  of  the  chromosomes, 


CRITICISMS    AND    CONCLUSIONS.  53 

although  Gerlach  (1906,  p.  13)  believes  that  the  typical  forms  appear  in 
the  prophase  only  and  that,  apparently  as  a  result  of  shrinkage,  the 
chromosomes  of  the  equatorial  plate  are  short,  rounded  rods,  like  those 
of  the  second  spindle.  This  supposed  change  of  form  is  explained  when 
it  is  noted  that  in  Gerlach 's  figures  the  chromosomes  of  the  first  spindle 
of  ovarian  eggs  (Gerlach  1906,  Taf.  i,  fig.  2,3)  have  the  typical  forms, 
while  the  oviducal  egg  (fig.  4)  with  supposed  first  spindle  has  the  rod- 
like  chromosomes;  for,  as  pointed  out  before,  what  he  calls  first  spindles 
in  oviducal  eggs  are  really  second  spindles.  Therefore,  Gerlach's  ma- 
terial, after  all,  presents  no  real  exception. 

Gerlach  (1906,  p.  25)  regards  the  chromosomes  of  the  first  spindle 
as  tetrads,  those  of  the  second  as  dyads.  The  conclusion  that  the  chro- 
mosomes of  the  first  spindle  are  tetrads  is  based  entirely  on  indirect 
evidence  and  on  reasoning  from  analogy  with  conditions  demonstrated 
in  many  invertebrates.  Since  in  the  first  polar  cell  he  finds  that  the 
chromosomes  sometimes  seem  to  be  present  as  dyads,  he  reasons  that 
those  of  the  first  maturation  spindle  must  have  been  tetrads. 

None  of  these  observers  has  recognized  and  figured  the  quadripar- 
tite structure  of  the  chromosome  of  the  first  maturation  spindle.  Both 
Tafani  and  Gerlach  (1906,  pp.  13-14),  it  is  true,  state  that  the  chromo- 
somes are  composed  of  Pfitzner's  granules  embedded  in  a  less  deeply 
stainable  substance ;  but  that  has  no  bearing  on  the  question  of  quadri- 
partite structure.  That  the  first  division  is  transverse  is  believed  by  all 
authors  except  Tafani  (1889,  p.  22),  who  thinks  it  longitudinal,  though 
he  has  not  directly  observed  it  in  the  mouse.  But,  since  he  confused 
the  two  spindles  with  each  other,  this  statement  applies  to  the  second 
spindle  only.  Sobotta  (1899,  1907)  alone  gives  illustrations  of  migrating 
daughter  chromosomes;  but  in  none  of  his  figures  does  he  show  their 
longitudinal  division.  There  is  no  doubt,  as  both  Sobotta  (1907,  p.  511) 
and  Kirkham  (19076,  p.  73)  state,  that  some  chromosomes  divide  earlier 
than  others. 

When  one  examines  carefully  the  accounts  of  the  first  maturation 
spindle  given  by  Sobotta  (1895,  I9°7)>  it  is  evident  that  in  his  first  paper 
he  speaks  of  a  relatively  early  stage  (fig.  40)  of  the  spindle  as  showing 
the  equatorial  plate,  a  stage  which  he  later  designates  correctly  as  the 
prophase.  Subsequent  writers — Gerlach,  Kirkham — have  figured  simi- 
lar stages,  and  Kirkham  (19076,  p.  73,  fig.  2)  has  applied  the  expression 
equatorial  plate  even  to  a  stage  in  which  the  chromosomes  are  distrib- 
uted over  half  the  length  of  the  spindle.  Gerlach  (1906,  p.  13),  however, 
clearly  states  it  as  his  opinion,  and  in  this  we  believe  he  is  right,  that 
such  spindles  are  still  in  process  of  formation;  but,  in  our  opinion,  he 
fell  into  an  error  in  ascribing  to  a  later  stage  of  the  first  spindle  a  condi- 
tion which  is  to  be  found  only  in  the  second  maturation  spindle;  for  he 
says  that  when  the  equatorial  plate  is  fully  formed  it  presents  in  the  side 
view  of  the  spindle  a  fairly  uniform  appearance,  its  chromosomes  having 


54     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

the  form  of  short  rounded  rods  such  as  Sobotta  shows  in  his  (1895) 
fig.  ioa.    But  Sobotta,  as  we  think,  and  as  he  would  probably  now  admit, 
made  a  mistake  in  supposing  that  his  figures  10  and  ioa  represented  the 
first  maturation  spindle.    The  egg  in  question  was  taken  from  the  ovi- 
duct, and  therefore  exhibits  the  second  maturation  spindle.     It  may  be 
noted,  in  passing,  that  by  some  strange  slip  of  the  pen  Sobotta  (1895, 
p.  91)  describes  his  fig.  loc  as  representing  the  beginning  of  metakinesis 
instead  of  an  advanced  anaphase.    In  his  more  recent  paper  he  (Sobotta, 
1907,  pp.  508-511,  fig.  2,  fig.  3)  has  figured  two  spindles  which  may 
more  properly  be  said  to  exhibit  an  equatorial  plate,  though  even  here 
the  chromosomes  do  not  assume  that  rigid,  plate-like  arrangement  which 
characterizes  the  equatorial  plate  in  many  other  animals  and  also  that 
of  the  second  maturation  spindle  in  the  mouse.     This  equatorial-plate, 
or  monaster,  stage  of  the  first  spindle  is  distinguished  (Sobotta,  1895, 
pp.   508-511)   from  the  prophase  by  the  possession  of  smoother  and 
straighter   spindle   fibers   and  by  the  predominance  of    chromosomes 
having  a  large  one-sided  protuberance.    There  is  no  disagreement  among 
authors  concerning  the  orientation  of  the  chromosomes  on  the  spindle 
nor  concerning  the  fact  that  they  vary  in  size.     But  as  to  the  number 
of  chromosomes,  there  is  a  wide  difference  of  opinion.     Tafani  and  the 
present  writers  count  20.     Sobotta — whose  view  has  been  accepted  by 
all  subsequent  investigators,  apparently  under  the  influence  of  the  large 
amount  of  his  material — maintained  in  1895  that  there  were  12  chromo- 
somes; but  recently,  stimulated  by  Dr.  J.  A.  Murray  to  a  reexamination 
of  his  material,  he  has  changed  his  opinion,  and  in  two  papers  (1907,  p. 
512;  1908,  pp.  248,  259)  has  stated  that  the  number  is  certainly  16. 
Holl  (1893,  P-  284)  argued  that  since  at  an  earlier  stage  there  were  24 
chromatic  balls,  there  should  be  as  many  loop-like  chromosomes,  and  was 
able  to  count  20;  but  not  much  weight  can  be  given  to  his  conclusions. 
He  admits  that  it  was  impossible  to  count  the  chromosomes  accurately. 
The  short  account  by  Melissinos  (1907,  p.  584)  is  remarkably  un- 
critical.   After  stating  that  Tafani  gave  the  number  as  20,  Holl  as  iS,1 
Sobotta  as  12,  and  others  as  24,  he  remarks  that  Sobotta's  counting  seems 
to  him  the  more  accurate,  and  then  proceeds  to  state  that  he  can  make 
out  only  8.     But  his  figures  are  too  diagrammatic  to  inspire  much  con- 
fidence on  the  part  of  the  reader. 

As  already  shown  (p.  45),  the  number  of  eggs  in  which  Sobotta  could 
possibly  have  counted  chromosomes  is  really  small.  In  1895  (p.  46)  he 
maintained  on  the  strength  of  many  successive  countings  of  the  same 
material  that  the  slender  (second)  spindle  in  all  probability  possessed 
12  chromosomes,  surely  not  over  14  or  15.  Moreover,  in  the  case  of  the 
thicker  first  spindle  (p.  51)  there  were  three  eggs  in  which  he  counted 

1  It  is  not  clear  how  Melissinos  comes  to  make  Holl  responsible  for  the  view 
that  the  mouse  egg  shows  18  chromosomes,  unless,  perchance,  his  eye  fell  on  the 
page  (280)  where  Holl  reports  that  Riickert  found  "about  18  chromatin  rods"  in 
Selachian  eggs. 


CRITICISMS    AND    CONCLUSIONS.  55 

"with  absolute  certainty'1  12  chromosomes,  and  in  many  other  instances 
approximately  12.  Now,  however,  apparently  without  any  additional 
material,  he  (1907,  p.  512;  1908,  p.  248)  counts  16!  Gerlach  (1906,  p.  23) 
expresses  himself  as  emphatically  agreeing  with  Sobotta  in  his  early 
statement  that  the  number  is  1 2 ,  he  (Gerlach)  having  repeatedly  counted 
12  in  both  the  first  and  the  second  spindle.  Lams  et  Doorme  count 
the  same  number,  12,  in  two  polar  cells;  but  we  have  shown  (p.  42)  that 
the  number  in  the  polar  cell  has  no  significance.  Kirkham  (19076,  pp. 
74-78)  likewise  affirms  that  there  are  12  chromosomes,  and  in  those  cases 
where  there  are  obviously  more  than  12  bodies  he  explains  the  higher 
number  as  being  due  to  the  precocious  division  of  some  of  the  chromo- 
somes. Nevertheless,  in  Kirkham's  own  preparations,  which  were  so 
generously  loaned  to  us,  out  of  four  normal  ovarian  eggs  in  the  stage  of 
the  first  spindle  there  were  three  cases  in  which  we  could  count  20  with 
certainty,  and  in  the  remaining  one  17. 

ACHROMATIN. 

Gerlach  (1906)  and  Sobotta  (1908,  p.  508)  are  the  only  writers  on 
the  maturation  of  the  egg  in  mice  who  give  any  opinion  as  to  the  precise 
origin  of  the  fibers  of  the  first  spindle.  These  they  think  arise  from  the 
linin  network  of  the  germinative  vesicle.  But  this  seems  improbable  in 
view  of  the  fact  that  there  is  a  stage  before  their  appearance  in  which 
only  shreds  of  the  linin  network  are  left,  while  most  of  the  vesicle  is  filled 
with  a  clear  fluid.  It  is  possible  that  the  linin  plays  some  part  in  the 
origin  of  the  spindle;  but,  as  has  already  been  suggested,  other  parts  of 
the  nucleus,  including  the  nucleolus,  are  the  more  probable  sources. 

Tafani  has  pointed  out  that  in  its  early  stages  the  first  spindle  in 
ovarian  eggs  is  short  and  fat,  a  condition  we  also  have  found.  Sobotta 
(1895,  1899,  1907)  figures  in  a  diagrammatic  way  the  spindle  with  sharp 
poles,  the  fibers  converging  to  a  point.  Lams  et  Doorme  (1907,  p.  274) 
say  the  fibers  converge  more  or  less  to  a  point.  Kirkham  figures  the 
shape  of  the  first  spindle  as  elliptical. 

According  to  Sobotta  (1907)  the  largest  spindle  is  30  to  32  micra 
long  and  20  micra  broad.  The  largest  spindles  we  have  found  have  the 
following  dimensions:  29.5  micra  in  length  by  n  in  breadth,  and  22.6 
in  length  by  14  in  breadth.  From  Sobotta's  paper  of  1899  it  must  be 
inferred  that  the  size  varies.  The  statement  of  Lams  et  Doorme  (1907, 
p.  275)  and  our  own  observations  accord  with  this  inference.  Gerlach's 
statement  (p.  10)  that  the  size  depends  in  the  main  on  the  size  of  the 
germinative  vesicle  can  not  be  accepted  as  demonstrated,  for  the  spindle 
is  not  a  result  of  the  metamorphosis  of  a  network  confined  in  a  rigid 
vesicle;  besides,  the  membrane  of  the  vesicle  has  nearly  disappeared 
when  the  spindle  is  first  differentiated. 

Sobotta  described  the  spindle  fibers  in  1895  (P-  51)  as  fine»  wavy, 
and  branched;  in  1907  (p.  508)  as  wavy  with  slight  thickenings.  His 
latter  description  applies  to  the  early  stages  of  the  first  spindle,  for  later 


56     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

the  fibers  become  thickened  at  the  polar  ends,  as  he  and  Lams  et  Doorme 
figure  them.  Gerlach  does  not  agree  with  Sobotta  that  there  is  a  central 
spindle.  While  we  have  no  evidence  of  the  existence  of  a  central  spindle 
like  that  discovered  by  Hermann,  we  agree  with  Sobotta  that  there  are 
some  fibers  which  run  from  pole  to  pole  without  being  attached  to  chro- 
mosomes. These  probably  persist  as  a  part  of  the  interzonal  filaments. 

CENTROSOMES,  CIRCUMPOLAR  BODIES,  AND  CLEAR  REGION. 

No  one  (with  the  possible  exception  of  Gerlach,  fig.  2)  has  figured 
the  corpuscles  near  the  poles  of  the  spindles  which  we  have  called  cir- 
cumpolar  bodies.  Tafani  (1889,  p.  22),  Sobotta  (1907,  p.  521,  for  the 
second  spindle  only),  and  Gerlach  (1906,  p.  9),  nevertheless,  mention 
granules  at  the  poles,  which,  according  to  the  two  latter  authors,  form  a 
sort  of  mantle  around  the  poles  of  the  spindle  and  thus  obscure  its  fibrous 
structure.  Gerlach  describes  them  as  occurring  with  both  spindles  and 
adds  that  they  sometimes  have  the  form  of  tortuous  threads,  which 
suggests  to  him  that  they  may  be  mitochondria. 

The  first  impression  one  forms  of  these  bodies  is  that  they  are  arti- 
facts due  to  improper  fixation;  but  when  one  reflects  that  they  occur 
in  eggs  fixed  by  different  methods  and  that  they  are  characteristic  of 
certain  stages  (see  p.  33),  this  interpretation  seems  unwarranted.  These 
bodies  were  also  seen  in  Kirkham's  preparations,  although  he  does  not 
himself  mention  them. 

A  study  of  the  occurrence  of  these  bodies  brings  out  the  fact  that 
they  are  characteristic  of  certain  periods  of  morphological  activity. 
For  example,  they  can  be  found  for  a  short  time  before  and  during  meta- 
kinesis  of  the  first  spindle  and  during  the  early  existence  of  the  second 
spindle  when  division  is  likely  to  occur  as  a  result  of  semination.  Con- 
versely, they  are  absent  during  periods  of  morphological  quiescence, 
such  as  the  telophase  of  both  spindles,  and  when  the  second  spindle 
persists  in  the  absence  of  semination.  It  will  be  remembered  that  these 
periods  of  activity  are  very  short  (p.  1 6),  while  the  quiescent  periods 
are  comparatively  long;  therefore  these  bodies  exist  during  only  brief 
periods.  The  question  naturally  arises,  Are  they  the  result  or  the  cause 
of  the  morphological  changes?  Unless  it  can  be  shown  that  they  are 
handed  on  from  cell  to  cell,  it  seems  reasonable  to  suppose  them  products 
rather  than  causes  of  spindle  activity.  On  the  other  hand,  the  absence 
of  typical  centrosomes  leads  one  to  ask  whether  they  may  not  in  some 
way  fulfill  the  function  of  centrosomes,  especially  since  they  are  situated 
very  close  to  the  poles  of  the  spindle.  Such  inquiries  can  not  be  answered 
at  present;  these  bodies,  the  existence  of  which  is  beyond  dispute,  are 
worthy  of  more  extensive  study,  and  their  possible  relation  to  mito- 
chondria should  certainly  be  investigated  further. 

Tafani,  Sobotta,  and  Gerlach  deny  the  regular  existence  of  centro- 
somes. Gerlach  (1906,  p.  26)  saw  in  one  case  two  centrioles  at  the  pole 
of  a  spindle,  and  Sobotta  (1907,  p.  524,  fig.  8)  figures  a  disk-shaped 


CRITICISMS    AND    CONCLUSIONS.  57 

body  at  one  pole  of  a  spindle,  where  a  centrosome  might  be  expected; 
but  he  declines  to  regard  it  as  such,  because  it  is  an  isolated  case.  Lams 
et  Doorme  (1907,  p.  274)  and  Kirkham  (19076,  p.  74)  alone  assert  the 
occasional  presence  of  these  structures,  the  former  saying  that  there 
are  usually  none  with  the  first  spindle.  Lams  et  Doorme  illustrate  two 
first  spindles  in  side  view,  one  in  an  egg  from  the  ovary  (fig.  2)  and  one 
from  the  oviduct  (fig.  5),  the  latter  being  the  case  to  which  Sobotta 
calls  attention  as  the  exception  to  the  rule  that  the  first  spindle  is  con- 
fined to  ovarian  eggs.  In  the  first  case  (fig.  2)  they  show  no  centrosomes, 
but  in  the  case  of  the  egg  from  the  oviduct  (fig.  5)  a  curved  rod  occupies 
one  pole  of  the  spindle.  The  latter,  however,  is  probably  a  second  spindle, 
since  the  egg  is  in  the  oviduct  and  since  all  the  second  spindles  figured 
by  them  have  somewhat  similar  centrosomes;  furthermore,  the  chromo- 
somes of  this  spindle  resemble  the  chromosomes  of  the  second  spindle 
rather  than  those  of  their  fig.  2 .  As  for  the  centrosomes  drawn  by  Kirk- 
ham,  their  presence  is  probably  referable  to  the  condition  of  the  eggs, 
many  of  which,  as  judged  from  an  examination  of  his  slides,  were  not 
normal.  It  will  be  noted  that  some  of  his  spindles  do  not  show  centro- 
somes; they,  we  believe,  are  normal.  There  seems,  then,  to  be  no  good 
ground  for  the  assertion  that  centrosomes  exist  in  connection  with  the 
first  spindle. 

Sobotta  (1895,  P-  44)  states  that  the  clear  region  around  the  chro- 
mosomes of  the  spindle  of  eggs  which  produce  only  one  polar  cell  has 
almost  precisely  the  extent  of  the  vanished  germinative  vesicle.  Since 
this  statement  really  relates  to  a  spindle  which  does  not  originate  from 
the  germinative  vesicle  directly  (as  Sobotta  himself  now  admits),  it 
loses  its  significance.  Lams  et  Doorme  (1907,  p.  274),  who  make  a 
similar  assertion  in  connection  with  the  first  spindle,  apparently  have 
not  themselves  seen  the  early  stages  (their  fig.  3  being  that  of  the  second 
spindle),  and  consequently  have  no  other  ground  than  Sobotta  for  their 
assertion.  According  to  our  descriptions  (pp.  26,  27,  33)  this  clear  region 
has  no  direct  relation  to  the  germinative  vesicle.  Since  it  exists,  as  the 
circumpolar  bodies  also  exist,  during  the  periods  of  morphological 
activity  of  the  spindle,  it  also  is  probably  a  manifestation  of  such  activity. 
POSITION  AND  ORIENTATION. 

Sobotta  (1895,  1899,  1907)  places  much  emphasis  on  the  position 
of  the  first  spindle,  which  is  situated  deep  in  the  egg.  Our  specimens 
substantially  corroborate  his  statement.  Regarding  the  angle  which  the 
axis  of  the  spindle  makes  with  the  surface  of  the  egg,  there  is  some  dis- 
agreement among  authors,  arising,  as  it  seems  to  us,  from  the  paucity 
of  proper  stages  in  the  material  which  most  of  the  investigators  have 
studied.  It  has  been  shown  (p.  33)  that  the  spindle  may  be  parallel  or 
oblique  to  the  surface,  but  that  it  is  only  rarely  perpendicular  at  any 
stage.  Tafani  (1889,  P-  22)  says  that  the  spindle  is  from  the  first  oblique, 
not  perpendicular,  and  figures  it  in  an  oblique  position  during  the  abstric- 


58     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

tion  of  the  polar  cell.  Although  the  statement  may  be  based  upon  the 
sscond  spindle,  which  Tafani  mistook  for  the  first,  it  nevertheless  is 
true  of  the  first  spindle.  Sobotta  (1895,  P-  48)  makes  the  unqualified 
statement  that  the  slender  spindle  (which  he  now  calls  the  second) 
turns  from  the  paratangential  position  to  the  oblique  and  finally  to  the 
radial  just  before  the  polar  cell  is  cut  off.  He  saw  three  cases  of  meta- 
phase  spindles,  all  oblique,  but  all  of  those  in  the  telophase  were  radial. 
Therefore,  although  he  had  not  actually  seen  the  process  of  abstriction, 
he  thought  the  first  spindle  was  radial  at  the  time  the  polar  cell  was 
cut  off.  In  a  later  paper  (1899,  p.  190)  he  describes  the  same  process 
for  the  first  spindle  and  gives  a  figure  (fig.  4)  of  the  spindle  during  the 
dyaster  stage  in  what  appears  to  be  a  radial  position  with  one  pole  in 
the  polar-cell  protrusion.  The  figure  has  a  somewhat  rigid  diagrammatic 
appearance  and  is  not  accompanied  by  any  explanation  to  prove  that  the 
spindle  is  radial  with  respect  to  the  center  of  the  egg  as  well  as  the  center 
of  the  section  in  which  it  lies.  The  relative  shortness  of  the  spindle 
suggests  the  possibility  that  its  axis  is  oblique  to  the  plane  of  the  section 
and  that  consequently  it  may  not  be  strictly  radial  in  position.  He 
mentions  having  three  other  spindles  in  the  stage  of  his  fig.  4,  but  does 
not  state  what  their  position  is.  In  one  of  his  recent  papers  Sobotta 
(1907,  p.  517)  figures  a  dyaster  stage  of  the  first  spindle  (fig.  4)  and 
states  that  it  is  in  an  oblique  position,  having  begun  the  rotation  from 
the  tangential  to  the  radial  position.  In  a  foot-note,  however,  he  admits 
that  it  really  is  never  met  with  in  a  strictly  radial  position!  He  (1907, 
p.  517)  finds  it  difficult  to  decide  whether  the  first  spindle  always  rotates, 
yet  he  argues  that  it  must  remain  tangential  in  most  cases  (one  polar 
cell)  because  it  is  transformed  in  the  monaster  condition  directly  into 
the  monaster  of  the  second  spindle,  which  is  likewise  tangential.  He 
is  not  sure  whether  even  in  one-fifth  of  the  cases  (those  in  which  it  divides) 
it  may  not  be  oblique  when  the  polar  cell  is  formed,  but  thinks  it  may  be 
assumed  that  as  a  rule  it  rotates,  because  the  second  spindle  always 
rotates,  and  because  it  (the  first)  takes  up  a  position  so  near  the  surface 
of  the  egg  that  no  polar  cell  could  be  produced  without  its  rotation. 
Sobotta  does  not  give  any  proof,  except  that  contained  in  his  first  paper 
(1895),  tnat  the  second  spindle  is  radial  at  the  moment  the  polar  cell 
is  abstricted.  Moreover,  he  figures  (1907,  fig.  9)  a  dyaster  of  the  second 
spindle  in  a  paratangential  position  and  says  (p.  525)  that  its  not  being 
radial  is  purely  accidental!  Thus,  except  for  his  1899  paper,  which  he 
does  not  mention  in  this  connection,  there  is  no  evidence  that  either 
polar  cell  is  cut  off  while  the  spindle  is  in  a  strictly  radial  position.  Ger- 
lach  (1906,  p.  10),  while  he  does  not  take  exception  to  the  general  conclu- 
sion of  Sobotta  that  there  is  a  rotation  of  the  spindle  from  a  tangential 
toward  a  radial  direction,  thinks  that  the  strictly  radial  position  is  not 
necessary  to  the  formation  of  the  polar  cell.  Neither  Gerlach,  Lams  et 
Doorme  (p.  275),  nor  Kirkham  (19076,  p.  75)  mention  having  seen  any 


CRITICISMS    AND    CONCLUSIONS.  59 

stages  of  the  metaphase,  and  the  latter  two,  having  seen  spindles  in 
oblique  positions,  apparently  assume  that  Sobotta  is  right  in  his  opinion 
that  the  spindle  becomes  radial  and  that  the  oblique  position  is  simply 
an  intermediate  one. 

DIVISION  OF  FIRST  SPINDLE  AND  ABSTRICTION  OF  FIRST  POLAR  CELL. 

Sobotta  (1899)  is  the  only  observer  who  has  figured  stages  in  the 
migration  of  the  daughter  chromosomes  towards  the  poles  of  the  spindle. 
Because  of  the  scarcity  of  such  stages  in  his  material  he  concludes  that 
the  first  spindle  divides  in  only  one-fifth  of  the  eggs.  In  the  other  four- 
fifths,  therefore,  the  spindle  does  not  divide  and  the  first  polar  cell  is 
not  cut  off.  This  may  possibly  be  due  to  a  failure  of  the  spindle  to  rotate 
(Sobotta,  1907,  p.  518,  footnote).  This  he  thinks  agrees  with  his  obser- 
vation that  80  per  cent  of  the  fertilized  eggs  have  only  one  polar  cell,  this 
one  being  in  his  opinion  the  equivalent  of  the  second  polar  cell  of  those 
eggs  which  form  two  such  cells. 

It  has  been  shown  (p.  16)  that  this  stage  is  of  very  short  duration. 
Hence  we  draw  the  conclusion  that  the  infrequent  occurrence  of  this 
stage  is  due,  not  to  the  failure  of  the  spindle  in  some  cases  to  divide,  but 
to  the  fact  that  the  chances  of  meeting  with  it  are  few. 

Gerlach  (1906,  fig.  5)  figures  a  recently  formed  polar  cell  in  an  ovarian 
egg,  but  he  says  nothing  about  the  division  of  the  supposed  first  spindle 
in  oviducal  eggs.  As  Sobotta  points  out,  supposed  first  spindles  in  the 
oviduct  have  had  as  much  time  in  which  to  divide  as  have  the  first  spin- 
dles of  adjacent  eggs  which  have  produced  the  first  polar  cells.  These 
considerations  go  to  show  that  Gerlach  misinterpreted  the  spindles  in 
oviducal  eggs. 

In  the  opinion  of  Sobotta  the  "Zwischenkorperchen,"  sometimes 
in  two  rows,  are  finally  inclosed  in  the  polar  cell  when  it  is  cut  off.  He 
describes  and  illustrates  this  condition  in  his  papers  of  1895  ano^  I9°7- 
Although  his  observations  were  really  made  on  the  second  spindle,  they 
hold  also  for  the  first.  It  is  difficult  to  account  for  this  conclusion  except 
on  the  ground  of  variable  conditions  or  poorly  preserved  material,  for, 
as  Lams  et  Doorme  (for  the  second  spindle)  and  Gerlach  show,  and  as 
our  material  so  clearly  proves,  the  bodies  in  question  do  not  lie  inside 
the  membrane  of  either  egg  or  polar  cell.  Gerlach,  however,  thinks  they 
are  at  first  in  two  rows  which  then  fuse. 

In  the  process  of  abstriction,  as  described  on  pp.  34  and  40,  there 
appears  to  be  an  attraction  between  the  "Zwischenkorperchen"  and 
the  vitelline  membrane.  Naturally  any  attraction  between  the  mem- 
brane and  these  bodies  would  be  exerted  more  readily  with  the  spindle 
in  an  oblique  or  tangential  position  and  its  effect  would  be  first  manifested 
on  the  side  of  the  spindle  nearest  the  surface.  It  is  perhaps  possible, 
then,  that  the  "Zwischenkorperchen"  have  some  part  to  play  in  the 
abstriction  of  the  polar  cell. 


60  THE    MATURATION    OF    THE    EGG    OF    THE    MOUSE. 

3.  SECOND  SPINDLE. 

CHROMATIN. 

It  was  Tafani  (1889,  P-  23)  wno  first  announced  that  in  the  greater 
number  of  cases  in  mice  only  a  single  polar  cell  is  formed.  It  was 
therefore  his  opinion  that  the  chromosomes  which  remained  in  the  egg 
after  the  formation  of  the  first  polar  cell  gave  rise  either  to  the  second 
spindle  (few  cases)  or  to  the  female  pronucleus  (greater  number  of  cases) . 
This  opinion  would  be  the  natural  consequence  of  his  probable  confusion 
of  the  second  spindle  with  the  first.  Sobotta  in  his  early  paper  (1895, 
p.  44)  also  held  that  in  those  eggs  which  produced  but  one  polar  cell 
(in  nine-tenths  of  the  cases,  in  his  opinion)  the  spindle  was  formed 
directly  from  the  germinative  vesicle,  and  (1895,  p.  53)  that  in  all  other 
eggs  (one-tenth  of  the  total  number)  the  second  spindle  was  produced 
from  the  chromosomes  which  remained  in  the  ovum  after  the  first  polar 
cell  was  abstricted.  Since  Sobotta  considered  the  spindle  in  the  former 
instance  to  be  the  equivalent  of  that  in  the  latter,  it  follows  that,  accord- 
ing to  his  view,  the  second  spindle  was  formed  in  some  cases  directly 
from  the  germinative  vesicle.  In  a  later  paper  (1907,  p.  514)  he  says 
that  he  has  no  observations  to  prove  this  view  and  that  it  is  erroneous. 
As  stated  in  this  paper  (1907,  p.  519),  he  now  believes  that  (hi  a  larger 
proportion, 'about  one-fifth  of  the  cases)  the  second  spindle  originates 
as  previously  described  for  one-tenth;  but  in  4  out  of  every  5  eggs  the 
monaster  of  the  second  spindle  is  derived  directly  from  the  monaster  of 
the  first,  i.e.,  without  the  formation  of  a  polar  cell.  That  is,  the  first 
spindle  in  a  large  proportion  of  ova  does  not  divide,  but,  in  some  way 
which  involves  a  degeneration  of  half  of  the  chromosomes  within  the 
cytoplasm  of  the  egg  (1907,  p.  541;  1908,  p.  250),  is  transformed  into 
the  corresponding  condition  of  the  second  spindle.  This  belief  he  thinks 
accords  with  his  observation  that  in  preserved  material  the  occurrence 
of  the  division  of  the  first  spindle  is  very  infrequent. 

This  is  Sobotta's  explanation  of  the  occurrence  of  only  one  polar 
cell  in  many  oviducal  eggs  in  the  late  stages  (the  ones  he  worked  with 
chiefly.  See  pp.  14,  45).  It  is  not  based  on  any  observation  of  degen- 
erating chromosomes  or  of  the  supposed  stages  of  transformation.  In 
fact,  Sobotta  repeatedly  says  that  he  has  seen  no  such  stage,  although  he 
believes  that  in  a  single  instance  (1907,  fig.  8,  a  spindle  with  more  than 
1 6  chromosomes,  which  occurred  in  an  oviducal  egg)  he  may  have  had 
an  example.  It  should  be  noted  that,  if  this  transformation  occurs  in 
four-fifths  of  all  the  eggs,  the  chances  of  meeting  with  it  must  be  four 
times  as  many  as  the  chances  of  encountering  the  division  of  the  first 
spindle.  In  view  of  these  considerations  one  may  be  warranted  in  ques- 
tioning the  existence  of  such  a  condition. 

Gerlach  (1906,  fig.  6)  illustrates  an  early  stage  in  the  origin  of  the 
second  spindle,  with  which  the  description  of  the  same  stage  in  the  pres- 
ent paper  agrees. 


CRITICISMS    AND    CONCLUSIONS.  6 1 

The  chromosomes  of  the  second  spindle  are  not  described  by 
Tafani,  except  as  the  description  which  he  gives  of  those  of  the  supposed 
first  spindle  really  applies  to  those  of  the  second.  Sobotta  (1907,  p.  521) 
holds  that  they  are  short  rounded  rods,  similar  in  form  to  the  daughter 
chromosomes  of  the  first  spindle,  though  generally  somewhat  smaller,  or 
at  least  slimmer.  Gerlach  (1906,  p.  14)  is  unable  to  distinguish  between 
the  chromosomes  of  the  first  and  second  spindles,  except  that  the  latter 
are  the  smaller;  he  figures  the  same  shapes  as  Sobotta,  and  also  a 
spindle  (fig.  16)  having  elongated  granular  chromosomes.  We  have 
found  in  many  spindles  in  which  the  chromosomes  are  closely  packed 
that  the  appearance — especially  of  those  chromosomes  which  are  seen 
in  end  view,  without  careful,  critical  study  and  comparison  with  more 
favorable  examples — seems  to  be  about  like  that  figured  by  Sobotta 
and  Gerlach.  Lams  et  Doorme  (1907,  p.  283)  think  that  the  presence 
of  the  first  polar  cell  is  the  only  reliable  criterion  for  identifying  the  second 
spindle.  Kirkham  (19076,  p.  78),  Sobotta  (1895,  P-  4-8),  and  Gerlach 
(1906,  p.  19)  state  that  the  daughter  chromosomes  elongate,  but  they 
describe  no  other  structure.  We  have  shown  this  lengthening  to  be 
characteristic  of  old  spindles. 

So  far  we  have  made  no  definite  statement  concerning  the  homol- 
ogies  of  the  chromosomes  of  the  second  spindle  with  those  of  the  first. 
Whether  the  mother  chromosomes  of  the  second  spindle  are  identical 
with  the  daughter  chromosomes  of  the  first  it  is  impossible  to  say  with 
certainty,  for  the  reason  that  there  is  no  way  of  determining  directly 
whether  or  not  the  chromosomes  which  become  fused  into  a  single  mass 
in  the  egg  after  the  first  polar  cell  is  cut  off  keep  their  individuality  and 
reappear  when  the  mass  breaks  up  preparatory  to  the  formation  of  the 
second  spindle.  The  striking  similarity  between  the  daughter  chromo- 
somes of  the  first  spindle  and  the  mother  chromosomes  of  the  second  in 
certain  cases,  and  also  analogy  with  those  invertebrates  in  which  the 
daughter  chromosomes  of  the  first  spindle  are  known  to  pass  directly 
to  the  second  spindle  without  undergoing  an  intervening  nuclear  or 
resting  stage,  make  it  seem  highly  probable  that  in  the  mouse  the  daugh- 
ter chromosomes  of  the  first  spindle  are  identical  with  the  mother  chro- 
mosomes of  the  second.  If  this  is  true,  then  the  division  between  the 
parts  of  the  chromosome  of  the  second  spindle  is  the  same  as  the  longi- 
tudinal division  in  the  daughter  chromosome  of  the  first  spindle  and  is 
therefore  apparent  in  the  fundaments.  On  this  ground  it  is  proper  to 
call  the  chromosomes  of  the  first  spindle  "tetrads,"  because  they  pos- 
sess the  two  divisions  which  mark  the  planes  of  separation  of  the  daughter 
chromosomes  of  two  quickly  ensuing  mitoses,  and  to  designate  those  of 
the  second  spindle  "dyads."  The  division  of  the  dyad,  then,  is  a  longi- 
tudinal splitting,  and  the  reduction  is  a  so-called  prereduction. 

Tafani  (1889)  makes  the  statement  that  the  chromosomes  of  the 
first  spindle  divide  longitudinally;  but,  as  we  have  seen,  this  statement 
5 


62     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

probably  relates  to  those  of  the  second  spindle.  Sobotta  (1895,  p.  46; 
1907,  p.  522)  and  Gerlach  (1906,  p.  14)  state  that  the  division  is  trans- 
verse, but  for  theoretical  reasons  they  believe  that  the  division  of  the 
chromosome  of  either  the  first  or  second  spindle  must  be  longitudinal. 
Sobotta  (1908,  fig.  7)  alone  figures  a  dividing  second  spindle.  His 
''biscuit"  shaped  chromosomes  remind  one  very  much  of  some  of  the 
dyads  we  have  described  (p.  37)  as  constituted  of  4  parts,  inasmuch  as 
the  "biscuit"  forms  are  in  some  instances  in  groups  of  4.  What  he  calls 
a  whole  chromosome  looks  more  like  half  of  a  dyad. 

The  same  criticisms  which  have  been  made  regarding  the  number 
of  chromosomes  of  the  first  spindle  apply  also  to  those  of  the  second.  It 
was  in  polar  views  (the  most  favorable  for  counting)  that  Tafani  found  20. 

ACHROMATIN. 

In  his  paper  of  1895  (p.  45)  Sobotta  stated  that  the  spindle  fibers 
of  the  single  spindle  (which  occurred  in  nine-tenths  of  the  eggs)  were 
derived  in  part  from  the  achromatic  portion  of  the  germinative  vesicle. 
As  already  pointed  out,  he  no  longer  holds  this  view. 

The  second  spindle  as  drawn  by  Sobotta  (1895, 1907) is  barrel-shaped, 
the  ends  being  somewhat  truncate,  the  fibers  only  slightly  curved,  and  the 
poles  open.  As  illustrated  by  Gerlach,  Kirkham,  and  the  present  writers, 
this  spindle  is  elliptical,  with  fibers  incurving  at  the  poles. 

The  flattening  of  some  of  the  second  spindles  described  on  page  38 
is  apparently  a  result  of  their  lying  close  to  the  surface  of  the  egg.  There 
is  a  possibility  that  the  flattening  is  caused  by  shrinkage  due  to  fixing 
and  dehydrating.  Shrinkage  to  produce  this  result  would  have  to  be 
greater  in  a  radial  than  in  other  directions,  and  could  be  explained  only 
on  the  supposition  that  the  substance  in  which  the  spindle  lies,  being 
probably  more  fluid  than  the  surrounding  cytoplasm,  is  extracted  more 
rapidly  on  the  side  nearest  to  the  surface  of  the  egg.  However,  were  the 
flattening  due  to  shrinkage  the  chromosomes  should  be  crowded  in  a 
radial  direction ;  but  that  this  crowding  does  not  exist  is  clear  from  plate 
4,  fig.  20,  in  which  the  spaces  between  the  chromosomes  are  as  uniform 
as  in  fig.  21. 

All  investigators  agree  that  the  second  spindle  is  smaller  than  the 
first.  Sobotta  (1907,  pp.  508,  520)  insists  that  the  second  is  but  half  the 
size  of  the  first,  although  he  does  not  state  whether  he  used  averages  for 
his  conclusion.  It  seems  unlikely  that  he  did,  since  he  says  that  his  fig.  3 
is  the  broadest  first  spindle.  It  must  be  admitted  that  a  first  spindle  may 
be  about  twice  the  size  of  a  second  spindle,  for  we  have  found  that  the 
largest  two  first  spindles  measure  29.5X11  micra,  and  22.6X14  micra, 
respectively,  and  the  smallest  second  spindles  14X6.5  micra  and  18X5.5 
micra,  respectively. 

All  who  have  published  papers  on  the  mouse,  except  Kirkham, 
figure  the  polar  ends  of  the  fibers  as  thickened.  In  regard  to  the  fibers 
which  are  not  attached  to  chromosomes,  there  is  no  conflict  between  the 


CRITICISMS    AND    CONCLUSIONS.  63 

statement  of  Sobotta,  that  there  are  fibers  stretching  from  pole  to  pole, 
and  our  own  results.  However,  he  gives  the  idea  that  such  fibers  form 
a  bundle  on  the  outside  of  which  the  chromosomes  rest  and  on  which 
they  are  drawn  to  the  ends  of  the  spindle,  whereas  the  distribution  of  the 
chromosomes  in  the  plane  of  the  equator  in  our  preparations  forces  us 
to  conclude  that  such  fibers,  if  present,  must  be  interspersed  among 
the  chromosomes.  Sobotta  (1895,  p.  47)  places  the  number  at  12  (later 
as  probably  16).  As  it  has  not  been  possible  to  count  them  in  our 
preparations,  we  can  not  state  what  the  number  is. 

CENTROSOMES,  CIRCUMPOLAR  BODIES,  AND  CLEAR  REGION. 

The  circumpolar  bodies  and  the  clear  region  have  already  been  con- 
sidered. The  former  dwindle  away  in  old  second  spindles,  leaving  what 
might  be  mistaken  for  centrosomes  (p.  39).  Such  remnants  may  well  be 
what  Sobotta  (1907)  and  Gerlach  (1906)  occasionaliy  saw  and  what  Lams 
et  Doorme  (1907,  figs.  6  to  8)  and  Kirkham  (1907)  found  more  regularly. 
Lams  et  Doorme  say  that  in  the  second  spindle  the  " centrosomes"  vary 
according  to  the  method  of  fixing.  But  in  our  opinion  these  are  not  to 
be  regarded  as  centrosomes. 

POSITION   AND  ORIENTATION. 

Gerlach  (1906,  pp.  18  to  20)  and  Kirkham  (19076,  p.  78)  have 
observed  that  the  second  spindle  or  second  polar  cell  may  be  at  various 
distances  from  the  first  polar  cell.  Sobotta  (1907,  p.  532)  finds  only  one 
such  condition  in  1,000  eggs  and  thinks  the  difference  between  Gerlach's 
material  and  his  may  be  due  to  the  fact  that  he  and  Gerlach  used  eggs 
of  different  ovulations.  We  are  at  a  loss  to  account  for  the  difference 
in  Sobotta's  material;  but  the  fact  nevertheless  remains  that  the  polar 
cells  may  be  found  at  various  distances  from  each  other.  Gerlach 
(1906,  pp.  1 8  to  20)  accounts  for  this  by  supposing  the  spindle  to  migrate 
through  the  cytoplasm,  and  he  figures  a  path  which  he  thinks  was  made 
by  such  a  moving  spindle.  The  distance,  he  believes,  is  determined  by 
the  epoch  of  semination,  because  with  that  event  the  second  spindle, 
wherever  it  may  be,  stops  in  its  migration  and  forms  the  second  polar 
cell  (or  at  least  divides) .  There  is  no  final  proof  that  this  migration  does 
not  occur,  but,  from  the  evidence  adduced  (p.  43)  in  connection  with 
the  position  of  the  first  polar  cell,  it  seems  simpler  and  more  reasonable 
to  suppose  that  the  polar  cell  shifts  its  position  under  the  zona.  This 
shifting  might  be  aided  by  the  power  the  polar  cell  has  of  changing  its 
shape,  as  was  observed  by  Tafani.  Such  an  explanation  makes  it  unnec- 
essary to  assume  changes  in  the  cytoplasm  and  a  migration  of  the  spindle 
that  is  so  out  of  harmony  with  what  is  known  in  other  animals,  where 
the  conditions  are  so  favorable  as  to  leave  no  doubt  as  to  the  events. 

The  orientation  of  the  second  spindle  is  like  that  of  the  first  and 
needs  no  further  discussion. 


64     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

DIVISION  OF  SECOND  SPINDLE  AND  ABSTRICTION  OF  SECOND  POLAR  CELL. 

The  only  illustrations  showing  the  division  of  the  second  spindle 
in  the  maturation  of  the  mouse  egg  are  those  of  Sobotta  (1895,  1907). 
The  criticisms  which  we  have  made  in  connection  with  the  division  of 
the  first  spindle  and  the  formation  of  the  first  polar  cell  (p.  59)  are  appli- 
cable to  the  corresponding  processes  in  the  second  oocyte. 

4.  POLAR  CELLS. 

There  is  agreement  among  the  investigators  of  the  mouse  egg  that 
not  all  fertilized  eggs  have  both  polar  cells.  According  to  Tafani  and 
Gerlach  the  first  polar  cell  is  always  formed,  but  the  second  in  a  large 
proportion  (respectively  four-fifths  and  three-fourths)  of  the  eggs  is  sup- 
pressed. Tafani  does  not  state  how  the  suppression  is  effected.  Gerlach 
thinks  that  in  the  event  of  late  semination  the  second  spindle  divides 
so  quickly  as  to  inhibit  the  formation  of  the  polar  cell  and  that  the  chro- 
mosomes which  would  have  been  contained  in  the  second  polar  cell 
remain  in  the  cytoplasm  of  the  egg  and  degenerate.  Although  he  avers 
that  he  has  seen  such  degenerating  chromatin,  it  should  be  borne  in 
mind  that  it  is  possible  he  mistook  for  chromosomes  cytoplasmic  bodies 
which  sometimes  stain  deeply  like  chromatin.  Sobotta,  on  the  other 
hand,  believes  that  in  most  cases  the  first  polar  cell  is  never  formed. 
In  1895  he  stated  that  even  the  first  spindle  did  not  come  into  existence. 
Now  (1907)  he  believes  that  the  spindle  is  formed  in  all  eggs,  but  that  in 
4  out  of  5  eggs  it  is  immediately  metamorphosed  into  the  second  spindle, 
half  of  the  chromatin  disintegrating  in  the  egg.  As  he  has  not  seen 
either  the  metamorphosis  or  the  degeneration  of  the  chromatin  he  has 
no  direct  evidence  for  his  belief.  Kirkham  states,  but  on  evidence  that 
in  part  at  least  is  unsound,  that  all  eggs  produce  the  first  polar  cell. 
His  explanation  (19076,  p.  80)  of  the  absence  of  one  polar  cell  is  appar- 
ently suggested  by  a  single  case  in  the  bat,  in  which,  according  to  van 
der  Stricht,  both  polar  cells  lay  outside  of  the  zona  pellucida.  It  is 
supported  by  one  observation  (Kirkham,  19076,  p.  81),  according  to 
which  the  polar  body  of  a  living  mouse  egg  (which  he  stained  and  dehy- 
drated under  the  microscope)  was  forced  through  the  zona  pellucida  by 
the  contraction  of  the  latter  under  the  influence  of  changing  osmotic  con- 
ditions. 

While  the  case  in  the  bat  is  suggestive  of  a  possible  explanation  for 
the  loss  of  the  first  polar  cell  in  the  mouse,  it  can  scarcely  be  admitted 
as  evidence  of  the  occurrence  of  such  conditions  in  the  mouse.  As  for  his 
observation  on  the  living  egg,  Kirkham  does  not  say  with  what  strength 
of  solutions  he  stained  and  dehydrated  the  egg  under  the  microscope. 
Although  he  may  have  seen  the  polar  cell  forced  through  the  zona  under 
direct  action  of  reagents,  the  same  thing  need  not  necessarily  occur  under 
natural  conditions,  since  eggs  in  the  oviduct,  and  still  more  those  in  the 
ovary,  are  protected  from  the  full  vigor  of  osmotic  action  by  the  sur- 


CRITICISMS    AND    CONCLUSIONS.  65 

rounding  fluid  in  the  oviduct  or  follicle  and  by  the  tissues  of  the  oviduct 
or  ovary.  Kirkham,  furthermore,  states  that  this  loss  of  the  polar  cells 
occurs  during  ovulation;  but,  since  he  has  not  seen  any  instances  in  which 
the  eggs  are  passing  from  the  follicles,  this  conclusion  must  be  based  on 
the  presence  of  these  bodies  at  one  stage  (viz,  before  ovulation)  and  their 
absence  at  another  (viz,  after  ovulation).  But,  unfortunately  for  this 
explanation,  they  are  not  universally  absent  in  the  latter  case. 

None  of  the  figures  of  mammalian  eggs  escaping  from  the  follicle— 
the  only  ones  known  to  us  being  those  given  by  Barry  (1839),  Sobotta 
(1895),  van  der  Stricht  (1901),  and  the  writers  (figs.  38,  39,  40) — furnishes 
any  evidence  whatever  that  the  polar  cell  is  being  pressed  through  the 
zona  pellucida.  Our  preparations  show,  on  the  contrary,  an  increased 
space  between  the  zona  pellucida  and  the  vitellus.  The  change  in  osmotic 
conditions  in  passing  from  the  ovary  to  the  periovarial  space  or  to  the 
oviduct  in  a  living  mouse  can  scarcely  be  great  enough  to  cause  the 
polar  cell  to  be  forced  through  the  zona  by  shrinkage  of  the  latter. 
Furthermore,  if  the  loss  of  the  first  polar  cell  is  caused  by  the  action  of 
reagents,  why  should  not  the  second  polar  cell  also  be  forced  through 
the  zona?  In  the  case  of  the  bat  van  der  Stricht  had  the  evidence  of 
both  polar  cells  lying  outside  the  zona.  There  is  not  even  this  evidence 
in  the  case  of  the  mouse,  for,  as  Sobotta  (1908,  p.  253)  has  observed,  no 
one  has  ever  seen  such  a  condition,  though,  if  it  occurs,  the  polar  cells 
should  be  easily  recognizable  among  the  surrounding  follicle  cells. 

There  is,  then,  no  good  evidence  of  the  suppression  of  either  polar 
cell  or  of  the  loss  of  the  first  polar  cell  by  extrusion  through  the  zona 
pellucida.  Lams  et  Doorme  (1907,  pp.  276,  287)  were  the  first  to  offer 
the  explanation  that,  while  both  polar  cells  are  formed,  the  first  under- 
goes degeneration  within  the  zona  and  disappears.  Their  figures  show 
this  clearly,  yet  they  suggest  that  what  they  call  degenerating  polar 
cells  may  possibly  be  bodies  (follicular  cells)  which  have  slipped  under 
the  zona!  Independently  of  Lams  et  Doorme,  and  before  their  paper 
was  published,  we,  also,  had  come  to  the  conclusion  that  the  first  polar 
cell  degenerates,  and  can  therefore  support  the  view  with  unbiased 
observations.  We  have  already  described  the  decrease  in  size  of  the  first 
polar  cell  and  the  evidence  of  the  degeneration  of  its  chromatin,  using 
polar  cells  of  eggs  which  contain  the  second  spindle  in  order  to  avoid 
even  the  possibility  of  confusing  the  first  polar  cell  with  the  second. 
Tafani  (1889,  p.  24)  mentions  that  the  first  polar  cells  vary  in  size  and 
also  calls  attention  to  cases  where  they  are  very  small.  Sobotta  (1907,  p. 
544)  alludes  to  these  small  forms  by  warning  his  readers  not  to  mistake 
for  polar  cells  what  he  says  may  be  follicle  cells  under  the  zona,  or  bodies 
formed  from  the  zona.  He  does  not  show  why  follicle  cells  should  be 
under  the  zona,  or  in  what  manner  they  could  get  into  such  a  position, 
or  how  the  zona  could  give  rise  to  bodies  with  nuclei.  It  must  be  remem- 
bered that,  since  Sobotta's  material  contained  a  large  proportion  of  the 


66     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

late  stages  (pronuclei  and  cleavage  stages) ,  it  presented  few  of  the  degen- 
erate polar  cells  (see  p.  41),  those  that  persisted  being  of  the  larger  size. 
Again,  eggs  fixed  in  osmic-acid  mixtures  (which  he  used  chiefly)  have  the 
zona  dark,  which  makes  it  difficult  and  often  impossible  to  interpret  or 
even  to  see  such  small  objects.  Upon  consideration,  it  is  not  surpris- 
ing that  the  first  polar  cell  should  degenerate,  for  usually  both  polar  cells 
do  so  in  time,  forming  no  part  of  the  embryo.  It  is  quite  possible  that 
the  substance  of  the  polar  cell  is  absorbed  by  the  egg. 

The  decrease  in  size  of  the  degenerating  polar  cell  explains  the  dis- 
agreement of  authors  concerning  the  relative  size  of  the  first  and  second 
polar  cells.  Sobotta  (1907,  p.  536)  maintains  that  sometimes  one,  some- 
times the  other,  is  larger.  Gerlach  (1906,  p.  13)  says  the  first  is  larger; 
Lams  et  Doorme  (1907,  p.  287)  that  the  second  is.  It  seems  fairly  cer- 
tain that  Lams  et  Doorme  must  have  seen  old  first  polar  cells  and  young 
second  ones,  for  they  have  few  of  the  earlier  stages,  even  though  they 
show  the  first  polar  cell  decreasing  in  size. 

Gerlach  (1906,  p.  25)  thought  that  in  one  first  polar  cell  the  chromo- 
somes were  dyads.  Sobotta  (1907,  p.  537)  says  that  both  polar  cells  may 
have  either  scattered  chromatin  or  a  nucleus,  which  is  formed  later  than 
the  egg  nucleus.  In  our  opinion  this  statement  must  mean  that  he  con- 
fused the  polar  cells,  for,  of  the  507  eggs  with  the  second  spindle  that 
we  have  studied,  none  have  a  first  polar  cell  with  a  nucleus;  whereas  the 
second,  in  seminated  eggs,  always  forms  a  nucleus  without  its  chromo- 
somes becoming  scattered  and  distinct.  Kirkham  (19076,  fig.  14),  also, 
has  probably  mistaken  the  first  polar  cell  for  the  second  in  the  figure  in 
which  he  shows  the  monads  much  separated. 

The  difference  in  chromatin  contents  of  the  two  polar  cells  accords 
with  the  well-known  fact  that  the  first  polar  cell  corresponds  to  the  first 
oocyte,  while  the  second  is  a  homologue  of  the  second  oocyte;  for,  on  the 
one  hand,  the  chromatin  of  the  first  polar  cell  does  not  form  a  resting 
nucleus, but  may  divide  (as  it  occasionally  does),  and,  on  the  other  hand, 
the  chromosomes  contained  in  the  second  polar  cell  immediately  become 
metamorphosed  into  a  nucleus  corresponding  to  the  egg  nucleus.  The 
first,  being  a  cell  which  degenerates,  divides  not  regularly  and  normally, 
but  with  what  seems  to  be  imperfect  mitosis  or  even  amitosis. 

5.  REDUCTION. 

It  is  fair  to  assume  from  the  preceding  account  that  the  longitudinal 
division  in  the  tetrads  corresponds  to  the  longitudinal  split  in  the  spireme 
of  a  synapsis  stage,  and  that  the  transverse  division  marks  the  place  of 
union,  end  to  end,  of  two  somatic  chromosomes.  Since  the  tetrad  gives 
rise  to  two  dyads  by  parting  along  the  transverse  plane  of  division,  and 
since  the  dyads  form  their  daughter  chromosomes  by  means  of  the  longi- 
tudinal division,  the  maturation  of  the  mouse  egg  belongs  to  the  class  of 
prereduction  divisions. 


SUMMARY    OF    PRINCIPAL    RESULTS.  67 

IX.  SUMMARY  OF  THE  PRINCIPAL  RESULTS  IN  THE  STUDY  OF 
THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

1.  Parturition  occurs  at  any  time  during  the  24  hours  of  a  day,  but 
more  frequently  in  the  early  morning. 

2.  The  stages  of  the  formation  of  the  first  spindle,  the  division  of  the 
first  spindle,  the  formation  of  the  second  spindle,  and  the  division  of  the 
second  spindle  are  relatively,  and  probably  absolutely,  very  short. 

3.  The  whole  maturation  process  requires  not  less  than  4  nor  more 
than  15  hours. 

4.  Maturation  usually  occurs  at  some  time  during  the  period  ex- 
tending from  13!  to  28  J  hours  after  parturition. 

5.  Ovulation  may  occur  at  any  time  during  a  period  beginning  at 
14^  and  ending  at  28^  hours  after  parturition. 

6.  Ovulation  may  occasionally  take  place  in  the  stage  of  the  first 
spindle,  sometimes  during  that  of  the  telophase  of  the  first  spindle  and 
the  formation  of  the  second  polar  cell,  but  usually  not  till  the  egg  con- 
tains the  second  spindle. 

7.  Insemination  is  most  successful  when  it  occurs  between  the  i8th 
and  3oth  hours  after  parturition. 

8.  The  spermatozoa  reach  the  egg  in  from  4  to  7  hours,  or  more, 
after  insemination. 

9.  The  pronuclei  are  formed  probably  within  a  few  minutes  after 
the  penetration  of  the  spermatozoon. 

i  o.  The  diameter  of  the  egg  decreases  from  the  stage  of  the  germina- 
tive  vesicle  until  it  reaches  the  oviduct,  when  it  increases  slightly. 

1 1 .  The  chromosomes  of  the  first  spindle  are  formed  from  the  chro- 
matin  of  the  germinative  vesicle,  and  possibly  also  from  the  wall  of  the 
nucleolus. 

12.  They  are  formed  before  the  nuclear  membrane  disappears. 

13.  They  show  indications  of  both  transverse  and  longitudinal  divi- 
sions, and  are  therefore  "tetrads." 

14.  In  the  first  maturation  division  the  tetrads  divide  transversely. 

15.  All  first  spindles  divide. 

1 6.  The  spindle  fibers  are  probably  derived  in  part  from  the  nu- 
cleolus. 

17.  The  chromosomes  of  the  second  spindle  are  "  dyads"  and  divide 
longitudinally,  separating  along  a  plane  which  is  probably  identical  with 
the  longitudinal  division-plane  of  the  tetrads. 

1 8.  The  chromosomes  of  each  spindle  number  twenty. 

19.  Typical  centrosomes  are  wanting  in  both  spindle  figures. 

20.  Bodies  surrounding  the  poles  of  the  spindles,  here  called  cir- 
cumpolar  bodies,  and  the  clear  region  surrounding  the  spindle  are  char- 
acteristic of  morphologically  active  stages  of  the  spindle. 


68     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

21.  Each  spindle  is  oblique  to  the  surface  of  the  egg  at  the  beginning 
of  the  abstriction  of  its  polar  cell. 

22.  All  eggs  form  two  spindles  and  a  first  polar  cell. 

23.  All  seminated  eggs  form  a  second  polar  cell. 

24.  The  first  polar  cell  probably  migrates  in  the  perivitelline  space 
inside  the  zona  pellucida,  and  is  aided  in  this  movement  by  the  process 
of  ovulation. 

25.  The  first  polar  cell  may  or  may  not  degenerate. 

26.  Maturation  division  in  the  mouse  egg  belongs  to  the  type  known 
as  prereduction  division. 


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EXPLANATION   OF  PLATES. 

All  drawings  were  made  with  the  aid  of  a  camera  lucida.  The  figures  as  repro- 
duced are  four-fifths  the  diameter  of  the  original  drawings.  The  magnification 
appended  to  the  description  of  each  figure  is  that  of  the  reduced  reproduction,  the 
magnification  of  the  original  drawing  being  in  parenthesis. 

The  magnification  of  2500  diameters  (reduced  =  2000)  was  obtained  with  a 
Zeiss  2mm.  homog.  immersion  apochromatic  objective  and  No.  12  compensating 
ocular;  that  of  1200  (reduced  =  9 60),  with  2mm.  objective  and  No.  6  compensat- 
ing ocular;  that  of  880  (reduced  =  7 04),  with  2mm.  objective  and  No.  4  compen- 
sating ocular;  and  that  of  170  (reduced  =  136),  with  Zeiss  A  objective  and  No.  4 
Huyghenian  eyepiece. 

PLATE  1. 

ORIGIN  OF  FIRST  MATURATION  SPINDLE. 

Fig.    i .  Germinative  vesicle  shortly  before  the  disappearance  of  its  nucleolus  and 
the  transformation  of  its  contents  into  the  fundaments  of  the  chromo- 
somes and  the  spindle  fibers.     Ovarian  egg.     X  (2500)  2000. 
Fig.     2.  Early  stage  in  the  formation  of  the  chromosome  fundaments.     Ovarian 

egg.      X(25oo)  2000. 

Figs.  2a,  2b.  Fundaments  of  chromosomes  in  sections  adjacent  to  that  of  fig.  2. 
Figs.  3 a,  36.  Two  consecutive  sections   showing  a  somewhat  later  stage  than  the 

preceding.     Ovarian  egg.      X  (2500)  2000. 

Figs.  4,  40.  Chromosomes  (20  in  number)  more  completely  differentiated.  Spindle 
not  yet  formed.  Nuclear  membrane  still  intact.  Ovarian  egg. 
X  (2500)2000. 

Fig.  5.  Section  of  a  young  spindle  showing  faint  fibrillations.  There  are  20  chro- 
mosomes scattered  over  its  surface.  Nuclear  membrane  is  dissolved 
at  some  points.  Ovarian  egg.  X  (2 500)  2000. 

Fig.  6.  Composite  drawing  of  a  spindle  cut  into  three  parts.  There  are  20  chromo- 
somes. Stage  slightly  more  advanced  than  that  illustrated  in  fig.  5. 
Nuclear  membrane  completely  vanished.  Ovarian  egg.  X  (2  500)  2000. 
Figs.  7,  70.  Two  consecutive  sections  of  a  spindle,  like  that  shown  in  fig.  6,  seen  in 
end  view.  There  are  20  chromosomes,  10  in  each  section.  The  cyto- 
plasm shows  faint  radiations  about  the  spindle.  Ovarian  egg. 
X  (2500)2000. 

PLATE  2. 

FIRST  MATURATION  SPINDLE. 
Fig.    8.  Ovarian  egg.      The  chromosomes  have  become  arranged  in  the  plane  of 

the  equator.      X  (880)  704. 
Figs.  8a,  86.   Enlarged  views  of  the  two  sections  into  which  the  spindle  in  fig.  8  is 

cut.    There  are  20  chromosomes.     X(25oo)  2000. 
Fig.    9.  Section  of  a  spindle  like  that  in  fig.  8.      X  (2500)  2000. 

Figs.  ioa,  i  ob.  The  two  sections  of  a  spindle  of  which  the  fibers  at  one  pole  converge 
to  a  point.    There  are  20  chromosomes.    Ovarian  egg.     X  (2500)2000. 
Fig.  ii.   Section  of  a  spindle  similar  to  the  preceding.    Ovarian  egg.     X  (2500)2000. 
Fig.  12.  Ovarian  egg.    The  polar  ends  of  the  spindle  fibers  are  becoming  thickened, 
and  the  clear  region  about  the  spindle  is  visible.     One  of  the  20  chro- 
mosomes (some  of  which  are  in  adjacent  sections)  has  been  displaced 
into  the  cytoplasm.     X  (1200)  960. 

Fig.  13.  Ovarian  egg.  The  circumpolar  bodies  are  formed  at  the  poles  of  the  spin- 
dle, and  the  clear  region  is  evident.  X  (1200)  960. 

Fig.  130.  More  highly  magnified  view  of  the  spindle  shown  in  fig.  13.   X(25oo)  2000. 
Fig.  136.  View  of  that  portion  of  the  spindle  seen  in  fig.  130  whicn  falls  in  the  fol- 
lowing section.     X(25oo)  2000. 

PLATE  3. 

DIVISION  OF  FIRST  SPINDLE  AND  ABSTRICTION  OF  FIRST  POLAR  CELL  (FIGURES 

14  TO  18,  INCLUSIVE). 

Fig.  14.  Ovarian  egg  containing  an  oblique  spindle.  Several  of  the  chromosomes 
have  already  divided.  Circumpolar  bodies  numerous  and  conspic- 
uous. X  (1200)960. 

Fig.     140.  One  chromosome  from  the  spindle  in  fig.  14. 

Figs.  150,  156.  An  oblique  spindle  in  two  consecutive  sections,  showing  the  mi- 
gration of  the  daughter  ^  chromosomes.  Ovarian  egg.  X  (2500)  2000. 

Figs.  i6a-i6d.  Four  consecutive  sections  of  a  spindle  similar  in  stage  of  division  to 
that  of  fig.  17.  See  fig.  H  (p.  34).  Ovarian  egg.  X(25oo)  2000. 


72     THE  MATURATION  OF  THE  EGG  OF  THE  MOUSE. 

PLATE  4. 
SECOND  MATURATION  SPINDLE  (FIGURES  190-236,  INCLUSIVE). 

Figs.  1 7 a,  1 76.  The  two  sections  show  a  spindle  in  a  more  advanced  stage  of  divi- 
sion than  that  in  figs.  150,  156.  The  abstriction  of  the  polar  cell  has 
begun  in  the  vicinity  of  the  "Zwischenkorperchen."  Ovarian  egg. 

X(2500)2000. 

Fig.  1 8.  Polar  cell  recently  abstricted.    Ovarian  egg.     X  (2 500)  2000. 

Figs.  190,  196.  Two  sections  of  an  oviducal  egg  showing  polar  cell  and  egg  nearly 

severed  from  each  other.     Prophase  of  second  spindle.      X(25oo) 

2000. 

Figs.  20,  21.  Polar  views  of  chromosomes  of  second  spindle.    Fig.  20  from  an  ovi- 
ducal egg.    Fig.  2 1  from  an  egg  in  periovarial  space.     X  (2  500)  2000. 
Fig.  22.  Side  view  of  second  spindle.    Large  first  polar  cell  on  nearly  opposite  side 

of  egg.    Oviducal  egg.    X  (1200)960. 
Figs.  230,  236.  Spindle  in  paratangential  position,  cut  obliquely  into  two  sections. 

There  are  19  chromosomes.     Circumpolar  bodies  not  stained  deeply. 

First  polar  cell  very  small  and   near   the  spindle.     Oviducal  egg. 

X  (2500)  2000. 

PLATE  5. 
SECOND  SPINDLE  AND  FORMATION  OP  SECOND  POLAR  CELL. 

Figs.  240,  246.  A  spindle  similar  to  that  of  fig.  23,  cut  into  two  parts.  There  are 
20  chromosomes.  First  polar  cell  absent.  Oviducal  egg.  X(25oo) 
2000. 

Figs.  25-27.  Old  second  spindles  from  three  eggs  showing  diminution  of  circum- 
polar  bodies.  All  from  oviducal  eggs  without  first  polar  cell.  X  (2500) 
2000. 

Figs.  280,  286.  Polar  views  of  the  two  daughter  plates  of  a  dividing  second  spindle 
in  a  stage  corresponding  to  that  in  fig.  16,  plate  3.  The  first  polar  cell 
is  very  small.  Oviducal  egg.  X(25oo)  2000. 

Figs.  290,  296.  Two  sections  of  an  oviducal  egg.  The  oblique  spindle  is  more  ad- 
vanced than  the  one  in  fig.  28.  The  stage  of  the  abstriction  of  the 
polar  cell  (see  also  fig.  /,  p.  40)  corresponds  to  that  of  figs.  16-17.  The 
first  polar  cell  is  seen  lying  at  the  left  of  the  second  in  fig.  296.  The 
egg  contains  the  heads  of  two  spermatozoa.  X(25oo)  2000. 

Fig.  30.  Oviducal  egg  showing  second  polar  cell  newly  abstricted,  the  first  polar 
cell,  and  the  head  of  a  spermatozoon.  X  (1200)  960. 

Figs.  310,  316.  Two  sections  of  an  egg  (another  section  of  which,  less  highly  mag- 
nified, is  shown  in  fig.  40)  exhibiting  in  fig.  310  the  first  polar  cell 
lying  in  the  enlarged  perivitelline  space.  X(i2oo)  960. 

PLATE  6. 

Figs.  32-37.  First  polar  cells  from  oviducal  eggs  which  contain  the  second  spindle. 
They  form  a  series  of  steps  which  illustrate  the  degeneration  of  the 
first  polar  cell.  Figs.  32  and  33  are  of  polar  cells  which  have  divided 
into  two  or  more  parts.  X(25oo)  2000. 

Figs.  38-40.  Three  stages  in  the  process  of  oyulation.  In  all  three  cases  the  egg  con- 
tains the  second  spindle  and  is  accompanied  by  the  first  polar  cell. 
X(i7o)  136. 

NOTE. — A  minute  body  appearing  in  the  clear  space  between  zona  pellu- 
cida  and  vitellus  in  fig.  38  is  due  to  a  defect  in  the  plate. 

PLATE  A. 

Fig.  A.  Mouse  cage.     For  description  see  p.  6. 

Figs.  B,  C.  Suspended  mouse  cages,  with  self-recording  apparatus  to  indicate  ap- 
proximately the  time  of  parturition  of  a  gravid  female.  For  descrip- 
tion see  pp.  7-10. 

Fig.  G.  Chromosomes  of  first  maturation  spindle.     See  pp.  28-30. 

(Plate  A  faces  page  6.) 


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